Activation mechanism and molecular engineering of Staphylococcus aureus Cas9

Activation mechanism and molecular engineering of Staphylococcus aureus Cas9 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Activation mechanism and molecular engineering of Staphylococcus aureus Cas9 Osamu Nureki, Satoshi Omura, Ryoya Nakagawa, Shohei Kajimoto, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7614961/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Apr, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Staphylococcus aureus Cas9 (SaCas9) is smaller than the widely used Streptococcus pyogenes Cas9 (SpCas9) and has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Furthermore, the precise nuclease activation mechanism of SaCas9 remains elusive. Here, we rationally engineered a SaCas9 variant (eSaCas9-NNG) with an expanded target scope and reduced off-target activity. The eSaCas9-NNG induced indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice. We further determined the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for the improved specificity and illuminating notable differences in the activation mechanisms between the small SaCas9 and the larger SpCas9. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biotechnology/Gene therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The RNA-guided DNA endonuclease Cas9 associates with a single-guide RNA (sgRNA) to cleave double-stranded DNA targets (dsDNA) complementary to the sgRNA guide. Since Streptococcus pyogenes Cas9 (SpCas9) exhibits high nuclease activity, it has been widely used for genome editing in eukaryotic cells 1 – 3 . Besides the guide RNA–target DNA complementarity, SpCas9 requires an NGG (where N is any nucleobase) sequence as the protospacer adjacent motif (PAM), restricting the targetable genomic sites 4 . To relax this constraint, we and others have engineered SpCas9 variants with altered PAM specificities, such as SpCas9-NG 5 and SpG/SpRY 6 . Although these SpCas9 variants enable genome editing at expanded target sites in various cell lines, their gene sizes (1,368 residues and 4.1 kb) pose a challenge in packaging them into an adeno-associated virus (AAV) vector for delivery into the target tissue, hampering their therapeutic applications 7 . Staphylococcus aureus Cas9 (SaCas9) consists of 1,053 residues (3.2 kb), approximately 0.95 kb shorter than SpCas9, and exhibits genome editing activities comparable to those of SpCas9 in human cells 8 . Notably, SaCas9, along with its sgRNA, can be packaged into a single AAV vector, enabling genome editing in mouse liver 8 . Furthermore, the catalytically inactive version of SaCas9 (dSaCas9) fused to a transcriptional regulator or the nickase version of SaCas9 (nSaCas9) fused to a cytosine or adenosine deaminase can be utilized as compact tools for transcriptional regulation or base editing, respectively 9 – 12 . However, SaCas9 requires relatively long NNGRRT (where R is A or G) PAMs, limiting its utility in genome editing applications 8 . Furthermore, the crystal structure of SaCas9 represents a catalytically inactive state, wherein the HNH nuclease domain is distant from the cleavage site in the target DNA strand (TS), likely due to the absence of the complementary non-target DNA strand (NTS) 9 . Therefore, the activation mechanism of SaCas9 remains enigmatic. Here, we rationally engineered a SaCas9 variant (eSaCas9-NNG) that recognizes relaxed NNG PAMs and exhibits reduced off-target cleavage, and demonstrated that eSaCas9-NNG can efficiently edit target sites with NNG PAMs in human cells and mice. We then determined the cryo-electron microscopy (cryo-EM) structures of eSaCas9-NNG in complex with its cognate sgRNA and dsDNA target in five distinct functional states, explaining how the introduced mutations alter the PAM specificity and enhance the cleavage fidelity. Our structures in multiple states reveal the stepwise domain rearrangements coupled to guide RNA–target DNA heteroduplex formation, highlighting the differences in the activation mechanisms between the small SaCas9 and the large SpCas9. Overall, this study demonstrates that the newly engineered SaCas9 variant can be harnessed as a compact and precise AAV-deliverable genome editing tool, and advances our understanding of the RNA-guided DNA cleavage mechanisms of the diverse Cas9 enzymes. Results Structure-guided engineering of the SaCas9-NNG variant To determine the optimal guide length for SaCas9, we performed in vitro cleavage experiments, using the purified SaCas9, sgRNAs with 20- to 23-nt guide segments (sgRNA20–23), and linearized plasmid DNA containing a target sequence and the canonical TTGAAT PAM (Supplementary Table S1 ). SaCas9 with all sgRNAs efficiently cleaved the DNA target, and sgRNA21 showed superior activity (Fig. 1 a and Extended Data Fig. 1 a), consistent with a previous study showing that 21-nt guide sgRNAs are optimal for SaCas9-mediated genome editing in human cells 8 . Therefore, we employed sgRNAs with 21-nt guides for the following experiments. To expand the targeting range of SaCas9, we sought to engineer a SaCas9 variant with relaxed recognition for the fourth to sixth positions in the NNGRRT PAM. Our previous structural analysis revealed that the third G in the PAM is recognized by Arg1015 in SaCas9, while the fourth and fifth Rs and the sixth T are recognized by Asn985 and Arg991, respectively 9 (Extended Data Fig. 1 b,c). We and others previously reported that PAM recognition can be relaxed by combining the elimination of base-specific interactions with PAM nucleotides and the introduction of non-base-specific backbone interactions with the PAM duplex 5 , 6 , 13 – 15 . Thus, we first purified the SaCas9 N985A/R991A variant, and measured its in vitro cleavage activity toward a target DNA bearing the TTGAAT PAM. As expected, the N985A/R991A variant showed almost no activity (Fig. 1 b). We then examined whether the N985A/R991A activity could be restored by replacing the residues surrounding the PAM duplex with basic or hydrophilic residues, and found that the E782K, L800R, T927K, N968R, and A1021S mutations partially restored the DNA cleavage activity toward the TTGAAT target (Fig. 1 b). The combination of all these mutations (N985A/R991A/E782K/L800R/T927K/N968R/A1021S; referred to as AAKRKRS) further enhanced the DNA cleavage activity (Fig. 1 b). However, the cleavage rate of AAKRKRS was still slightly lower than that of wild-type SaCas9 (referred to as SaCas9 for simplicity) (Extended Data Fig. 1 d). Molecular modeling suggested that the K929N and I1017F mutations form hydrogen-bonding and van der Waals interactions with Lys927 (T927K) and Arg1015, respectively, thereby stabilizing the interactions with the PAM duplex (Extended Data Fig. 1 e,f). Indeed, the inclusion of these two mutations into the AAKRKRS variant enhanced the cleavage activity toward the TTGAAT target to a level comparable to that of SaCas9 (Extended Data Fig. 1 d). To investigate whether the AAKRKNRFS (N985A/R991A/E782K/L800R/T927K/K929N/N968R/I1017F/A1021S) variant exhibits relaxed PAM recognition, we assessed the cleavage activities of SaCas9 and AAKRKNRFS toward target DNAs with 19 different PAMs, including TTG NN T and TTGAA N PAMs. SaCas9 efficiently cleaved the target DNAs with TTGRRN PAMs and showed a slight preference for T at the sixth position (Fig. 1 c), consistent with the previous study 8 . In contrast, AAKRKNRFS efficiently cleaved all target DNAs except for the TTGGTT target (Extended Data Fig. 1 g). The SaCas9 structure suggested a steric clash between the side chain of Asn986 and the methyl group of the fifth T, which could reduce the activity of AAKRKNRFS toward the TTGGTT target (Extended Data Fig. 1 h). Indeed, the addition of the N986S mutation (N985A/R991A/E782K/L800R/T927K/K929N/N968R/N986S/I1017F/A1021S; referred to as AAKRKNRSFS) enhanced the cleavage activity toward the TTGGTT target, although this variant still showed relatively lower activities for TTGGNN PAMs (Fig. 1 d). To comprehensively explore the PAM specificities of AAKRKNRSFS, we performed in vitro PAM discovery assays, using a DNA library containing the target sequence adjacent to a randomized 8-bp sequence. We confirmed that, whereas SaCas9 is specific to NNGRR PAMs, AAKRKNRSFS recognizes simple NNG sequences as the PAMs (Fig. 1 e). These results demonstrated that the engineered AAKRKNRSFS variant exhibits a relaxed PAM constraint, and thus we refer to it as SaCas9-NNG. A previous study reported the SaCas9-KKH variant (E782K/N968K/R1015H), which was engineered via directed evolution and recognizes relaxed NNNRRT PAMs 16 . To compare the PAM preferences between SaCas9-NNG and SaCas9-KKH, we evaluated their in vitro cleavage activities toward target DNAs bearing TT N AAT PAMs. SaCas9-NNG exhibited no activity except for the TTGAAT target, whereas SaCas9-KKH showed activity against all the PAMs (Fig. 1 f), consistent with the reported relaxed preference of SaCas9-KKH for the third G in the PAM. Nonetheless, under our assay conditions (50 nM Cas9), SaCas9-NNG was much more active than SaCas9-KKH toward the TTGAAT target (Fig. 1 f), demonstrating the superiority of SaCas9-NNG for targeting the NNG PAM targets. Genome editing by SaCas9-NNG in mammalian cells To assess the genome editing activity of SaCas9-NNG, we measured indel (insertions or deletions) formations induced by SaCas9 and SaCas9-NNG at 35 endogenous target sites with NNG PAMs in human embryonic kidney (HEK) 293T cells (Supplementary Table S2 ). As expected, SaCas9 induced indels at the NNGRR, but not NNGYY (where Y is T or C), target sites (Fig. 2 a). In contrast, SaCas9-NNG efficiently induced indels at the NNGRR sites (except for the NNGGG sites) and the NNGYY sites (Fig. 2 a), consistent with our in vitro cleavage data. We also measured the genome-editing efficiencies of SaCas9 and SaCas9-NNG in murine immortalized liver (TLR3) cells. SaCas9 efficiently edited only NNGRR sites, whereas SaCas9-NNG modified all NNG sites, albeit with lower activity at the NNGG sites (Extended Data Fig. 2 a). These results demonstrated that SaCas9-NNG can edit target sites with NNG PAMs in mammalian cells, although with relatively reduced activity at NNGG PAM targets. Base editing by SaCas9-NNG in human cells Next, we examined whether SaCas9-NNG can be harnessed for base-editing technology. We designed the D10A nickase versions of SaCas9 and SaCas9-NNG fused to the activation-induced cytidine deaminase (referred to as SaCas9-AID and SaCas9-NNG-AID, respectively), as in the SpCas9-based cytidine base editor 17 , and measured C-to-T conversion efficiencies at 35 endogenous target sites with NNG PAMs (identical to those tested for indel formation) in HEK293T cells (Supplementary Table S2 ). SaCas9-AID efficiently mediated C-to-T conversions at the NNGRR, but not NNGYY, target sites (Fig. 2 b). In contrast, SaCas9-NNG-AID showed C-to-T base conversions toward all the target sites, albeit with lower efficiencies at the NNGG sites (Fig. 2 b). Furthermore, we designed the nickase versions of SaCas9 and SaCas9-NNG fused to TadA-8e (referred to as SaCas9-ABE8e and SaCas9-NNG-ABE8e, respectively), as in the SpCas9-based adenine base editor 18 – 20 , and measured A-to-G conversions toward various point mutations known to cause hemophilia B 21 in HEK293 cells (Supplementary Table S2 ). SaCas9-ABE8e exhibited A-to-G conversions only at NNGRR targets with relatively low efficiencies, whereas SaCas9-NNG-ABE8e induced A-to-G conversions at all targets (Extended Data Fig. 2 b,c). A-to-G base conversions predominantly occurred between the 7th and 20th positions from the PAM within the protospacer (Extended Data Fig. 2 b,c), largely consistent with the previously reported target window of SaCas9-ABEmax 11 , 12 . These results demonstrated that the catalytically inactive version of SaCas9-NNG can serve as a useful RNA-guided DNA-targeting platform. Genome editing by SaCas9-NNG in mice Since SaCas9 (1,053 residues) is smaller than SpCas9 (1,368 residues), the SaCas9-NNG gene, along with its sgRNA and/or accessory components, can be packaged into an all-in-one AAV vector, enabling its genome-editing applications in living organisms. We designed a single AAV vector, encoding SaCas9 or SaCas9-NNG under the HCRhAAT promoter and a U6 promoter-driven sgRNA targeting the F9 gene for hemophilia B (Fig. 2 c). We injected 7-week-old mice with 1×10 12 vector genomes (vg) of the single AAV serotype 8 vector, and measured the indel formation at 12 weeks after the injection. SaCas9 induced indels at the NNGGAT and NNGAAA, but not NNGCAA and NNGTCA, target sites (Fig. 2 d). In contrast, SaCas9-NNG efficiently induced indels at all the targets (Fig. 2 d). Consistently, SaCas9-mediated editing of the F9 target with the NNGGAT and NNGAAA PAMs reduced the plasma coagulation factor IX (FIX) activity, whereas SaCas9-NNG-mediated editing at all target sites attenuated the FIX activity (Fig. 2 e). These results indicated that SaCas9-NNG exhibits the expanded target scope in mice, and could be used as a therapeutic genome-editing tool deliverable via a single AAV vector. Structure-guided engineering of the eSaCas9-NNG variant In addition to the limited target ranges due to the PAM requirement, off-target effects pose an obstacle to therapeutic applications of CRISPR-based technologies 22 – 24 . To reduce off-target cleavage by SaCas9, we sought to engineer a high-fidelity SaCas9 variant. Previous studies revealed that reducing non-specific interactions between SpCas9 and the DNA backbone improves cleavage fidelity 25 – 28 . In the SaCas9 structure, Asn413 interacts with the ribose moiety of dC19 in the target DNA 9 (Extended Data Fig. 3 a). Indeed, the N413A mutation did not alter the on-target activity in vitro , but reduced the cleavage activity against an off-target DNA containing a single mismatch at the PAM distal end (Fig. 3 a). A previous study also revealed that disrupting the salt bridges within the REC domain enhances the fidelity of SpCas9 28 . As observed in SpCas9, the Ala substitution of Arg420, which forms salt bridges with Glu406 and Asp412, reduced off-target cleavage by SaCas9, while maintaining its on-target activity (Fig. 3 a and Extended Data Fig. 3 a). Notably, the N413A/R420A double mutations further enhanced the fidelity of SaCas9, and the inclusion of these mutations also substantially reduced the off-target activity of SaCas9-NNG (Fig. 3 a). The N413A/R420A mutations reduced the cleavage kinetics of both SaCas9 and SaCas9-NNG toward the on-target DNA substrate, as also observed in high-fidelity SpCas9 variants 29 (Extended Data Fig. 3 b). To comprehensively assess the effect of the N413A/R420A double mutations on the target specificity, we examined the in vitro DNA cleavage activities of SaCas9 and SaCas9-NNG toward DNA substrates containing a mismatch at positions 1–21. While SaCas9 and SaCas9-NNG were tolerant to most single mismatches, the N413A/R420A mutations reduced the activities of SaCas9 and SaCas9-NNG toward mismatch-containing substrates, especially those with PAM-distal mismatches (positions 16, 17, 20, and 21) (Fig. 3 b). Thus, we refer to these high-fidelity variants as enhanced-specificity SaCas9 (eSaCas9) and eSaCas9-NNG, respectively. Furthermore, we compared the on- and off-target activities of eSaCas9 with those of SaCas9-HF (R245A/N413A/N419A/R654A), a rationally engineered high-fidelity SaCas9 variant 30 . Both variants exhibited similar on- and off-target activities (Fig. 3 b), demonstrating that eSaCas9 possesses comparable cleavage specificity to SaCas9-HF. Genome editing by eSaCas9 and eSaCas9-NNG in human cells We compared the genome-editing efficiencies of eSaCas9 and eSaCas9-NNG with those of SaCas9 and SaCas9-NNG at 35 endogenous target sites with NNG PAMs in HEK293T cells. Consistent with our in vitro data, eSaCas9 and eSaCas9-NNG exhibited comparable genome-editing efficiencies to those of SaCas9 and SaCas9-NNG, respectively (Fig. 3 c). Using GUIDE-seq (genome-wide, unbiased identification of double-stranded breaks enabled by sequencing), we analyzed the genome-wide specificities of SaCas9, eSaCas9, SaCas9-NNG, and eSaCas9-NNG at the VEGFA site in human cells. As expected, eSaCas9 and eSaCas9-NNG exhibited lower off-target activity than SaCas9 and SaCas9-NNG, respectively (Extended Data Fig. 3 c,d). These results demonstrated that the N413A/R420A mutation substantially reduces off-target cleavage by SaCas9, while maintaining on-target activity. To evaluate the advantages of eSaCas9-NNG over other Cas9 variants with relaxed PAM constraints, we compared the genome-editing efficiencies of eSaCas9-NNG with those of SpRY 6 , SpG 6 , and iGeoCas9 14,15 , which recognize NN, NG, and NNNNC as the PAMs, respectively, at six different target sites with NNG PAMs in HEK293T cells (Supplementary Table S2 ). eSaCas9-NNG and SpRY induced indels at the six target sites with 14.3% and 8.8% efficiencies on average, respectively, indicating that eSaCas9-NNG outperforms SpRY at target sites with NNG PAMs (Fig. 3 d,e). As expected, eSaCas9-NNG, but neither SpG nor iGeoCas9, efficiently induced indels at target sites with NTGNW PAMs (where W is A or T) (Fig. 3 d,e). Moreover, even for targets with NGGNC PAMs, which are equally compatible with the four Cas9 nucleases, eSaCas9-NNG exhibited genome-editing efficiencies comparable to or higher than those of SpG and iGeoCas9 (Fig. 3 d,e). Collectively, these findings establish eSaCas9-NNG as a versatile genome-editing tool that can be used for precise gene therapy with a broad target range and robust genome-editing activities. Cryo-EM structure of the eSaCas9-NNG–guide RNA–target DNA complex To elucidate the molecular mechanism underlying the relaxed PAM recognition and improved fidelity of eSaCas9-NNG, we determined the cryo-EM structure of eSaCas9-NNG in complex with a 98-nt sgRNA (containing a 21-nt guide) and its target dsDNA containing the TTGCCT PAM at 3.1-Å resolution (Extended Data Figs. 4 and 5 , and Supplementary Table S3,4). eSaCas9-NNG adopts a bilobed architecture consisting of recognition (REC) and nuclease (NUC) lobes, which are connected by a bridge helix (BH) and a linker loop (Fig. 4 a and Extended Data Fig. 5 a–c). The NUC lobe consists of the RuvC, HNH, WED, and PI domains, while the REC lobe consists of the REC1 and REC2 domains. Within the NUC lobe, the HNH and RuvC domains are connected by the L1 and L2 linkers. The sgRNA guide segment base-pairs with the TS to form a 21-bp guide RNA–target DNA heteroduplex, which is accommodated between the REC and NUC lobes (Fig. 4 a and Extended Data Fig. 5 b–e). The sgRNA scaffold comprises a repeat:anti-repeat duplex, stem-loop 1, and stem-loop 2 (Extended Data Fig. 5 d,e). The repeat:anti-repeat duplex is sandwiched between the REC1 and WED domains, while stem-loop 1 and stem-loop 2 are recognized by the BH/REC1 and RuvC/PI domains, respectively (Extended Data Fig. 5 c–e). Nucleotides dG(− 21*)–dA(− 17*) and dG(− 3*)–dC(− 1*) in the single-stranded NTS are bound to the positively charged surfaces of the RuvC and RuvC/L2/PI domains, respectively, while nucleotides dA(− 16*)–dT(− 4*) are disordered (Extended Data Fig. 5 c–e). The PAM-containing DNA duplex binds to the surface formed by the WED and PI domains, while nucleotides dA(− 25*)–dC(− 22*) in the NTS re-hybridize with nucleotides dG22–dT25 in the TS to form the PAM-distal DNA duplex (Fig. 4 a and Extended Data Fig. 5 c–e). Except for the HNH domain, eSaCas9-NNG is structurally similar to the previously reported crystal structure of SaCas9 9 , suggesting that the introduced mutations do not substantially affect the complex structure (Extended Data Fig. 6 a,b). In the SaCas9 crystal structure, the HNH domain is distant from the cleavage site of the TS and interacts with the RuvC domain, indicating that it represents the catalytically inactive state. In contrast, the HNH domain in the present structure docks onto the TS cleavage site and interacts with the REC1 domain and the L1 linker (Extended Data Fig. 6 a,b), and thus represents the catalytically activated state. Indeed, the TS was cleaved between dC3 and dA4 (Extended Data Fig. 6 c,d), although it contains phosphorothioate modifications, possibly due to the high enzyme concentration during reconstitution. The 3′-hydroxy group of dC3 and the phosphate group of dA4 are stabilized by a Mg 2+ ion, which is coordinated by Asp556 and Asn580 in the HNH domain (Extended Data Fig. 6 c,d). In addition, the dA4 phosphate group is recognized by the catalytic residue His557, corresponding to His840 of SpCas9 (Extended Data Fig. 6 c,d). As in the SaCas9 structure 9 , Asp10, Glu477, and His701 in the RuvC domain coordinate two Mg 2+ ions and form the active site responsible for the NTS cleavage (Extended Data Fig. 6 c,e). These structural observations demonstrated that eSaCas9-NNG cleaves the TS and NTS in Mg 2+ -dependent manners, using the HNH and RuvC nuclease domains, respectively, as observed in other Cas9 orthologs 31 – 33 . Structural basis for the relaxed PAM recognition by eSaCas9-NNG The TTGCCT PAM is recognized by the WED and PI domains in the eSaCas9-NNG structure (Fig. 4 a and Extended Data Fig. 6 f). As in the SaCas9 structure 9 , the third G in the PAM (dG3*) forms bidentate hydrogen bonds with the side chain of Arg1015, which is stabilized by interactions with Glu993 and Phe1017 (I1017F) (Fig. 4 a). By contrast, the fourth to sixth nucleobases in the PAM (dC4*–dT6*) lack base-specific interactions with the protein, due to the Ala985 (N985A), Ser986 (N986S), and Ala991 (R991A) replacements (Fig. 4 a). Notably, the newly incorporated Lys782 (E782K), Lys927 (T927K), and Arg968 (N968R) residues directly interact with the backbone phosphates of the PAM duplex, while Arg800 (L800R) and Ser1021 (A1021S) likely form water-mediated hydrogen bonds with the PAM duplex (Fig. 4 a). In addition, Asn929 (K929N) stabilizes the conformation of Lys927, which interacts with the backbone phosphate of dG3* (Fig. 4 a). Accordingly, these newly formed non-base-specific interactions compensate for the loss of base-specific interactions with the fourth to sixth PAM nucleobases, thereby achieving the relaxed NNG PAM recognition by SaCas9-NNG. Whereas Arg1015 hydrogen bonds with the fourth G in the NNGG PAMs in the SaCas9 structure 9 (Extended Data Fig. 6 g), Arg1015 is slightly farther away from the fourth PAM nucleobase in the eSaCas9-NNG structure, due to the presence of the introduced Phe1017, preventing the interaction between Arg1015 and the fourth G (Extended Data Fig. 6 g). These structural observations account for the reduced activity of eSaCas9-NNG toward DNA substrates with NNGG PAMs. Structural basis for the improved specificity of eSaCas9-NNG To understand the structural basis for the improved specificity of eSaCas9-NNG, we determined the cryo-EM structures of SaCas9 and eSaCas9-NNG in complex with an sgRNA and its target dsDNA containing a single-nucleotide mismatch at position 21 from the PAM (Fig. 4 b,c, and Extended Data Fig. 7). SaCas9 and eSaCas9-NNG adopt almost identical overall structures, indicating that the N413A/R420A mutations do not alter the overall conformation of the protein when bound to a mismatched target (Fig. 4 d). Notably, both structures represent the catalytically active conformation where the HNH domain docks onto the TS cleavage site, suggesting that the N413A/R420A mutations in the REC2 domain do not directly regulate the HNH domain conformation in the presence of a PAM-distal mismatch, in contrast to the allosteric regulation between the REC3 and HNH domains observed in high-fidelity variants of SpCas9 27 . The PAM-distal G1:dG21 mismatch likely forms a Hoogsteen base pair in both SaCas9 and eSaCas9-NNG, resulting in the slight distortion of the TS backbone (Fig. 4 e,f and Extended Data Fig. 8a,b). While the overall structures of SaCas9 and eSaCas9-NNG are similar, we observed a conformational difference in a helix of the REC2 domain (residues 414–421, referred to as the mismatch-sensing helix (MSH)) (Fig. 4 e–g and Extended Data Fig. 8c,d). In the SaCas9 structure, the MSH interacts with the PAM-distal TS backbone, with Asn413 and Asn419 forming hydrogen-bonding interactions with dC19 and dC20, respectively. Notably, Arg420 forms salt bridges with Glu406 and Asp412, stabilizing the relative position of the MSH. These interactions facilitate the stable binding of the mismatch-containing distorted TS to SaCas9, thereby enabling the efficient cleavage of a target DNA with a PAM-distal mismatch. In contrast, in the eSaCas9-NNG structure, the Asn413–dC19 hydrogen bond and the Glu406–Arg420–Asp412 salt bridge network are eliminated due to the N413A and R420A mutations, leading to an outward displacement of the MSH from the TS backbone. Furthermore, in the eSaCas9-NNG structure, the side chain of Asn419 adopts a flipped-out conformation and disrupts the Asn419–dC20 hydrogen bond. These local structural changes cause further distortion of the PAM-distal TS backbone, likely destabilizing the active-state conformation of the protein and impairing the ability of eSaCas9-NNG to efficiently cleave DNA with a PAM-distal mismatch (Supplementary Movie S1). Collectively, these structural observations suggest that the target specificity of SaCas9 is regulated by the interactions between the MSH and the PAM-distal TS backbone. Cryo-EM structures of eSaCas9-NNG in distinct functional states In addition to the catalytically active state (State I), our cryo-EM analysis revealed three distinct classes (States II–IV), which are primarily distinguished by the guide RNA–target DNA heteroduplex lengths (Extended Data Fig. 4 ). We therefore determined the three additional structures at overall resolutions of 3.2 Å (State II), 2.9 Å (State III), and 2.8 Å (State IV) (Fig. 5 a–c and Extended Data Fig. 4 ). In State II, the sgRNA guide does not hybridize with the double-stranded target DNA, which instead binds to the groove between the WED and PI domains, with the third G PAM nucleotide recognized by Arg1015 in the PI domain, as in the catalytically active state (Fig. 5 a,d). Thus, this structure likely represents the “interrogation state”, in which the Cas9–sgRNA complex recognizes a PAM sequence but has not yet unwound the double-stranded target DNA to hybridize with the TS, as previously observed in the cryo-EM structure of SpCas9 in the interrogation state, which was artificially stabilized by a protein–DNA cross-link 34 . In SpCas9 and SaCas9, the WED, RuvC, HNH, and PI domains are structurally ordered in this state and adopt similar conformations to those in the inactive state 34 , 35 . In contrast, the BH, REC1, and REC2 domains are disordered in the interrogation state of SaCas9, suggesting that they are flexible before the heteroduplex formation (Fig. 5 a). Notably, nucleotides G15–C21 in the sgRNA are pre-ordered in an A-form geometry for base-pairing with the TS, even without interactions with the BH (Fig. 5 a). In State III, nucleotides A4–C21 in the sgRNA base-pair with nucleotides dG1–dT18 in the TS to form an 18-bp heteroduplex, while the three PAM-distal nucleotides (G1–G3) in the sgRNA are disordered (Fig. 5 b). This structure represents the “intermediate state”, in which the Cas9–sgRNA complex has partially formed the guide–target heteroduplex. In this state, the BH and the REC1 domain become ordered and extensively interact with the PAM-proximal region of the heteroduplex (Fig. 5 e). In contrast, the REC2 domain remains disordered, with the PAM-distal region of the heteroduplex exposed to the solvent (Fig. 5 b). The HNH domain is located far from the TS cleavage site, while the RuvC active site is occluded by the L1 linker, as in the interrogation state (Fig. 5 a,b), indicating that the formation of the 18-bp heteroduplex is insufficient to activate SaCas9, as previously reported 8 . In State IV, nucleotides G1–C21 in the sgRNA base-pair with nucleotides dG1–dC21 in the TS to form the complete 21-bp heteroduplex, as in the catalytically active state (Fig. 5 c). In this state, the REC2 domain becomes ordered and interacts with the PAM-distal region of the heteroduplex, while the HNH domain, along with the L1 and L2 linkers, becomes disordered (Fig. 5 c). Thus, this structure represents a “translocation state”, in which the HNH domain is moving toward the TS cleavage site. Although the dissociation of the L1 linker allows the NTS to access the RuvC domain, unlike the catalytically active state, only one Mg 2+ ion is bound to the RuvC active site (Fig. 5 f), indicating that the RuvC domain does not adopt a cleavage-competent active conformation in the translocation state. Nuclease activation mechanism These four structures in different functional states provide mechanistic insights into the dynamic nuclease activation of SaCas9. A structural comparison of the interrogation and intermediate states reveals that the ordering of the BH and the REC1 domain is coupled with the shift of the pre-ordered guide region (G15–C21) toward the interior of the protein, resulting in the formation of the 18-bp guide–target heteroduplex (Fig. 5 a,b). A structural comparison between the intermediate and translocation states demonstrates that the ordering of the REC2 domain is coupled with the formation of the 21-bp guide–target heteroduplex (Fig. 5 b,c). The heteroduplex elongation facilitates a conformational change in the L2 linker (residues 629–649) (Extended Data Fig. 9a–f). In the intermediate state, residues 635–645 in the L2 linker form an α helix and interact with both the RuvC and HNH domains, stabilizing the inactive conformation (Extended Data Fig. 9a,d). In contrast, in the translocation state, this α helix is structurally melted and the HNH domain becomes disordered (Extended Data Fig. 9b,e). A structural comparison of the two states suggests a steric clash between the L2 α helix in the intermediate state and the heteroduplex in the translocation state (Extended Data Fig. 9c,f), indicating that the formation of the 21-bp heteroduplex induces the structural rearrangement of the L2 linker, resulting in the dissociation of the HNH domain from the RuvC domain (Extended Data Fig. 9a–f). In the catalytically active state, the HNH domain undergoes an approximately 160° rotation from its inactive position and docks onto the TS cleavage site in the heteroduplex (Extended Data Fig. 9g,h). This HNH rearrangement is accompanied by structural changes in the REC1 domain and the L1/L2 linkers. Whereas residues 126–146 in the REC1 domain are located near the TS cleavage site in the intermediate and translocation states, these residues become disordered in the active state, due to the docking of the HNH domain (Extended Data Fig. 9h). The L1 linker undergoes an approximately 180° rotation from its position in the intermediate state and binds to the minor groove of the PAM-distal heteroduplex (Extended Data Fig. 9i,j). This L1 rearrangement is accompanied by a structural change in the β4 strand (Glu477) of the RuvC domain, thus forming the RuvC active site (Extended Data Fig. 9k). In the active state, the L2 linker adopts a loop conformation to form an NTS-binding pathway toward the RuvC active site (Extended Data Fig. 9j). In particular, Phe635 in the L2 linker stacks with the dA(− 1)-dT1* base pair in the PAM duplex, while Arg1002 in the PI domain interacts with the flipped-out dC(− 1*) in the NTS (Extended Data Fig. 9l). Collectively, these structural observations revealed the coordinated domain rearrangements coupled with the formation of the guide–target heteroduplex to achieve target DNA cleavage by SaCas9 (Supplementary movies S2 and S3). Discussion SaCas9 was identified in 2015 as the first compact Cas9, and has since been used as a versatile genome editing tool in human cells and various organisms 8 , 36 , 37 . However, the development of useful SaCas9 variants has been limited as compared to SpCas9, and the action mechanism of the small SaCas9 has remained enigmatic. In this study, we rationally engineered the eSaCas9-NNG variant with an expanded targeting range and reduced off-target activity. We also determined the cryo-EM structures of eSaCas9-NNG in four distinct states, illuminating the dynamic activation mechanism of the small SaCas9 enzyme. Recent studies revealed coordinated conformational rearrangements of multiple domains, including the REC2/REC3 and HNH domains, coupled with the guide–target heteroduplex formation in target DNA cleavage by SpCas9 31,33,34,38 . However, Cas9 orthologs have structurally diverse REC lobes, and small Cas9 enzymes, such as SaCas9, lack a domain equivalent to the REC2 domain of SpCas9 (the REC2 domain of SaCas9 corresponds to the REC3 domain of SpCas9). Therefore, the activation mechanisms of small Cas9 orthologs have remained elusive. The eSaCas9-NNG structures in the four different states illuminated the conformational rearrangements coupled with the guide–target heteroduplex formation, highlighting the mechanistic conservation of Cas9 nuclease activation. In the inactive states of both SpCas9 and SaCas9, the HNH domain is located far from the TS cleavage site (Extended Data Fig. 10a,b). Upon the guide–target heteroduplex formation and the accompanying REC rearrangement, the HNH domains of SpCas9 and SaCas9 undergo rotations of approximately 140° and 160°, respectively, docking onto the TS cleavage site (Extended Data Fig. 10c,d). In both SpCas9 and SaCas9, the L1 and L2 linkers participate in recognizing the PAM-distal region of the heteroduplex and guiding the NTS toward the RuvC active site, respectively (Extended Data Fig. 10e,f). These structural observations suggest that the inactive-to-active HNH domain rearrangements via the L1/L2 linkers are conserved among Cas9 activation mechanisms. We also found notable differences in the activation mechanisms between SpCas9 and SaCas9 (Fig. 6 ). In SpCas9, the entire REC lobe is ordered when the 6-bp guide–target heteroduplex forms, creating a positively charged cleft that accommodates the PAM-distal DNA duplex 33 . By contrast, in SaCas9, while the REC1 domain is ordered, the REC2 domain remains disordered even when the 18-bp guide–target heteroduplex forms. This difference is likely attributed to the absence of REC1–REC2 interactions and the presence of the larger WED domain in SaCas9, as compared to SpCas9. Furthermore, whereas the formation of a 12-bp heteroduplex induces a structural change in the HNH domain in SpCas9 33 , the formation of an 18-bp heteroduplex in SaCas9 does not induce the HNH rearrangement. This variation is likely due to the structural differences in the HNH domains and the L1/L2 linkers between SpCas9 and SaCas9. Collectively, the structurally divergent REC lobes and L1/L2 linkers contribute to the distinct activation mechanisms of the Cas9 orthologs, which may result in their different cleavage efficiencies and fidelities. Moreover, our cryo-EM structures revealed that the structurally divergent REC lobes also contribute to the distinct mismatch-sensing mechanisms of the Cas9 orthologs. In SpCas9, the REC3 domain senses the integrity of the PAM-distal region of the guide–target heteroduplex and allosterically regulates the HNH domain movement by reorienting the REC2 domain 27 , 39 . These allosteric domain rearrangements establish a conformational checkpoint that traps SpCas9 in an inactive state when bound to a mismatched target. Indeed, single molecule experiments have demonstrated that HNH domain docking onto the TS is completely abolished when some high-fidelity SpCas9 variants bind to mismatched targets 27 . In contrast, SaCas9 lacks REC1–REC2 interactions and employs a distinct mismatch-sensing mechanism, wherein the MSH in the REC2 domain detects PAM-distal mismatches and presumably affects the stability of the active conformation of the HNH domain, thereby governing its target specificity. While SpCas9 efficiently cleaves its target DNA using a 20-nt guide sgRNA, most small Cas9 orthologs require 1–2-nt longer guides for efficient DNA cleavage ( e.g. , 21- and 22-nt guides are optimal for SaCas9 and CjCas9, respectively) 1 , 8 , 40 , 41 . Although the choice of optimal guide lengths is an important factor in Cas9-mediated genome engineering, it remains unclear why the Cas9 orthologs require different guide lengths for efficient DNA cleavage. A structural comparison between SpCas9 and SaCas9 provides an explanation for their different optimal guide lengths. In the active state of SpCas9, Arg765 in the L1 linker interacts with the backbone phosphate of the 5′ end (G1) of the 20-nt guide 38 (Extended Data Fig. 10g). In the SaCas9 structure, Arg480 and Lys482 in the L1 linker interact with the backbone phosphate of G1 in the 21-nt guide (Extended Data Fig. 10h). These interactions between Cas9s and the 5′ ends of their guides are consistent with their optimal guide lengths. Although further elongation of the guide–target heteroduplex seems possible without steric clashes, the PAM-proximal end of the heteroduplex is fixed within the central groove of SaCas9, likely resulting in an energetically unfavorable supercoiling effect with extended base pairing outside the SaCas9 complex. In summary, we developed the eSaCas9-NNG variant, which will expand the CRISPR toolbox for in vivo therapeutic genome editing. In addition, our cryo-EM analysis provides structural snapshots of the small SaCas9 during target DNA cleavage, improving our mechanistic understanding of diverse CRISPR-Cas9 enzymes. To date, structure-guided Cas9 engineering has primarily relied on crystal structures captured in the inactive state. By leveraging active- and intermediate-state structures, such as those resolved in this study, future Cas9 engineering efforts can be guided by a more rational and precise structural framework, enabling the development of next-generation genome editors with enhanced specificity and efficiency. Methods Protein and RNA preparation for structural analysis The gene encoding full-length SaCas9 (residues 1–1,053) was codon optimized, synthesized (Genscript), and cloned between the Nde I and Xho I sites of the modified pE-SUMO vector (LifeSensors). The mutations were introduced by a PCR-based method, using the vector encoding full-length SaCas9 as the template, and the sequences were confirmed by DNA sequencing (Supplementary Table S2 ). The SaCas9 protein was expressed and purified using the protocol reported previously 42 . Briefly, the N-terminally His 6 -tagged SaCas9 proteins were expressed in Escherichia coli Rosetta2 (DE3). The SaCas9-expressing E. coli cells were cultured at 37°C in LB medium (containing 20 mg/L kanamycin) until the OD 600 reached 0.8, and protein expression was then induced by the addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside (Nacalai Tesque). The E. coli cells were further cultured at 20°C for 18 hr, and harvested by centrifugation at 5,000 g for 10 min. The E. coli cells were resuspended in buffer A (20 mM Tris-HCl, pH 8.0, 20 mM imidazole, and 1 M NaCl), lysed by sonication, and then centrifuged at 10,000 g for 20 min. The supernatant was mixed with 0.3 mL Ni-NTA Superflow resin (QIAGEN) equilibrated with buffer A, and the mixture was loaded into a Poly-Prep Column (Bio-Rad). The protein was eluted with buffer B (20 mM Tris-HCl, pH 8.0, 300 mM imidazole, and 300 mM NaCl). To remove the His 6 -SUMO-tag, the eluted protein was mixed with SUMO protease, and then dialyzed at 4°C overnight against buffer C (20 mM Tris-HCl, pH 8.0, and 300 mM NaCl). The protein was loaded onto a HiTrap SP HP column (GE Healthcare) equilibrated with buffer C, and eluted with a linear gradient of 0.3–2 M NaCl. The protein was further purified by chromatography on a HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated with buffer D (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 2 mM MgCl 2 , and 1 mM DTT). The purified proteins were stored at − 80°C until use. The sgRNA was transcribed in vitro with T7 RNA polymerase, using a partially double-stranded DNA template. The transcribed sgRNA was purified by 8% denaturing urea PAGE, extracted from gel slices with Tris-Borate-EDTA Buffer (Takara), and then ethanol precipitated. The sgRNA pellet was dissolved in nuclease-free water and stored at − 20°C. In vitro cleavage assay The linearized pUC119 plasmid (100 ng, 4.7 nM), containing the 23-nt target sequence and the PAMs (Supplementary Table S1 ), was incubated at 37°C for 0.5–5 min with the SaCas9–sgRNA complex (50 nM) in 10 µL of reaction buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 2 mM MgCl 2 , 1 mM DTT, and 5% glycerol). The reactions were stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng). The reaction products were resolved, visualized, and quantified with a MultiNA microchip electrophoresis device (SHIMADZU). PAM identification assay The PAM identification assay was performed as described previously 5 . The PAM library (100 ng), containing eight randomized nucleotides downstream of a 21-nt target sequence, was incubated at 37°C for 5 min with the purified SaCas9 (SaCas9 and SaCas9-NNG) (50 nM) and the sgRNA (21-nt guide) in 10 µL of reaction buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 2 mM MgCl 2 , 1 mM DTT, and 5% glycerol). The reactions were stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng), and then purified using a Wizard DNA Clean-Up System (Promega). The purified DNA samples were amplified for 25 cycles, using primers containing common adapter sequences. After column purification, each PCR product (~ 5 ng) was subjected to a second round of PCR for 15 cycles, to add custom Illumina TruSeq adapters and sample indices. The sequencing libraries were quantified by qPCR (KAPA Biosystems), and then subjected to paired-end sequencing on a MiSeq sequencer (Illumina) with 20% PhiX spike-in (Illumina). The sequencing reads were demultiplexed by primer sequences and sample indices, using NCBI Blast + (version 2.8.1) with the blastn-short option. For each sequencing sample, the number of reads for every possible 8-nt PAM sequence pattern (4 8 = 65,536 patterns in total) was counted and normalized by the total number of reads in each sample. For a given PAM sequence, the enrichment score was calculated as log 2 -fold enrichment as compared to the untreated sample. PAM sequences with enrichment scores of − 2.0 or less were used to generate the sequence logo representation, using WebLogo (version 3.7.1) 43 . The cumulative distribution and histogram of the read count of each PAM in the unedited sample confirmed that the plasmid library has sufficient coverage for the individual PAM sequences. Genome- and base-editing analyses in human cells Genome- and base-editing analyses were performed, according to the protocol described previously 44 . Briefly, HEK293Ta cells were maintained in DMEM (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Sigma), at 37°C in a 0.05% CO 2 atmosphere. HEK239Ta cells were seeded at 5×10 3 cells per well in collagen I-coated 96-well plates, 24 hr prior to transfection. HEK239Ta cells were transfected with a SaCas9 plasmid or a SaCas9-derived base-editor plasmid (120 ng) and an sgRNA plasmid (40 ng), using Polyethylenimine Max (Polysciences) (1 mg/mL, 0.5 µL) in PBS (50 µL) (Supplementary Table S2 ). The cells were harvested 3 days after transfection, treated with 50 mM NaOH (100 µL), incubated at 95°C for 10 min, and then neutralized with 1 M Tris-HCl, pH 8.0 (10 µL). The obtained genomic DNA was subjected to two rounds of PCR, to prepare the library for high-throughput amplicon sequencing. Genomic regions targeted by sgRNAs were PCR-amplified to add custom primer-landing sequences. The PCR products were purified by AMPure XP magnetic beads (Agencourt), and then subjected to a second round of PCR to attach the custom Illumina TruSeq adapters with sample indices. After size-selection by agarose gel electrophoresis and column purification, the sequencing libraries were quantified using a KAPA Library Quantification Kit Illumina (KAPA Biosystems), multiplexed, and subjected to paired-end sequencing (600 cycles), using a MiSeq sequencer (Illumina) with 20% PhiX spike-in (Illumina). The sequencing reads were demultiplexed, based on sample indices and primer sequences. Using NCBI BLAST + (version 2.6.0) with the blastn-short option, the sequencing reads were mapped to the reference sequences to identify indels and substitutions in the target regions. To remove common PCR errors and somatic mutations, we deleted sequencing reads containing mutations (> 1% frequency) commonly observed in the control samples from the edited samples, and then normalized the editing frequencies for the target sites by subtracting the mutation frequencies of the control samples from those of the edited samples. Comparison of genome-editing efficiencies among various Cas9 variants in human cells HEK293T cells were maintained in DMEM (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS) (Nichirei), 1% GlutaMAX (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Sigma), at 37°C in a 0.05% CO 2 atmosphere. Cells (5×10 4 cells/well) were seeded in 48 well plates coated with collagen type I (Cellmatrix type I-C, Nitta Gelatin) the day before transduction. The plasmids (200 ng) were incubated together with Lipofectamine 3000 (Thermo Fisher Scientific), and then directly added to the cell culture according to the manufacturer's recommendations. At 48 h after the transduction, the cells were lysed with the SimplePrep reagent for DNA (Takara Bio). The supernatants were directly used for PCR. DNA fragments were amplified with Phusion DNA polymerase (New England Biolabs). PCR amplicons were subjected to 150-bp pair-end read sequencing using the Illumina MiSeq at Genome-Lead Co., Ltd. (Kagawa, Japan). The frequencies of the mutations were assessed by CRISPResso2 45 . GUIDE-Seq analysis HEK293 and U2OS cells were maintained and cultured as described previously 46 . Cells were nucleofected following the manufacturer’s instructions (Lonza) in 20 µL Solution SE, using programs CM-104 (293) and DN-100 (U2OS) on a Lonza Nucleofector 4-D. Cells were transfected with SaCas9 plasmids (500 ng), sgRNA plasmids (250 ng), and dsODN [100 pmol; complementary oligonucleotide sequences derived from Malinin et al. 2021 47 ]. GUIDE-seq library preparation and analysis were performed as previously described 46 , 47 . Briefly, genomic DNA was purified via an Agencourt DNAdvance kit (Beckman Coulter). A Covaris E220 ultrasonicator was used to shear purified genomic DNA to an average fragment size of 500 bp. After sonication, 400 ng was used for library preparation. Genomic DNA was treated with end-repair mix (Qiagen), A-tailed with Taq polymerase (Thermo Fisher Scientific), ligated to single-tailed sequencing adapters, and purified using SPRI magnetic beads. Two rounds of nested PCR with the dsODN sense- and antisense-specific primers in separate reactions were performed on the adapter-ligated library. After purification with SPRI magnetic beads, libraries were quantified using the Kapa qPCR Library Quantification Kit (Kapa). Equimolar amounts of samples were pooled and sequenced with 150 bp paired end reads on the Illumina NextSeq 550 sequencer. For GUIDE-seq-2 library preparation, Tn5 transposase was prepared by combining hyperactive Tn5 with annealed i5 adapter oligos containing an 8-nucleotide barcode and 10-nucleotide unique molecular index, in 2x Tn5 dialysis buffer (100 mM HEPES-KOH pH 7.2, 200 mM NaCl, 0.2 mM EDTA, 2 mM DTT, 0.2% Triton X-100, and 20% glycerol), for one hr at 24°C. Tagmentation was performed in 40 µL reactions for 7 min at 55°C, using 250 ng of genomic DNA, 4 µL of assembled Tn5/i5-transposome, and 8 µL of fresh 5x TAPS-DMF buffer (50 mM TAPS-NaOH, 25 mM MgCl 2 , and 50% dimethylformamide (DMF)). To stop the reaction, 5 µL of a 50% proteinase K (NEB) solution was added, and the solution was incubated for 15 min at 55°C. Samples were purified using SPRI-guanidine magnetic beads, and separate PCR reactions were performed using dsODN sense- and antisense-specific primers. Reactions were conducted with Platinum Taq (Thermo Fisher) using the following thermocycler settings: 95°C for 5 min, 15 cycles of temperature cycling (95°C for 30 sec, 70°C (-1°C per cycle) for 120 sec, and 72°C for 30 sec), 20 constant cycles (95°C for 30 sec, 55°C for 60 sec, and 72°C for 30 sec), and 72°C for 5 min. PCR products were purified using SPRI beads and quantified using a Kapa qPCR Library Quantification Kit (Kapa). Libraries were purified using Lightbench (Yourgene Health) selection, and sequenced using a NextSeq1000/2000 (Illumina) sequencer with cycle settings of 146, 8, 18, and 146. Data analysis was performed using the updated open-source GUIDE-seq2 analysis software ( https://github.com/tsailabSJ/guideseq/tree/V2 ). Plasmid construction, AAV production, and assessment of genome editing AAVpro293T cells (Takara Bio) and HEK293 cells were cultured in DMEM (Sigma) supplemented with 10% FBS (Thermo Fisher Scientific) and GlutaMAX (Thermo Fisher Scientific). Murine immortalized liver cells (TLR3 cells, JCRB Cell Bank) were maintained in DMEM containing 2% FBS, 5 ng/mL of human epidermal growth factor (EGF), and ITS-X Supplement (Thermo Fisher Scientific). The SaCas9 cDNA was codon-optimized in GenScript. A DNA fragment comprising a promoter, SaCas9 cDNA (SaCas9 or SaCas9-NNG), the SV40 polyadenylation signal, and sgRNA sequence driven by the U6 promoter was introduced into the p1.1c plasmid. The HCRhAAT liver-tropic promoter (an enhancer element of the hepatic control region of the ApoE/C1 gene and the human anti-trypsin promoter) was employed. SaCas9-ABE8e and the human coagulation factor IX (FIX) cDNA were introduced into the pcDNA3 (Thermo Fisher Scientific) and pBApo-EF1α Neo (Takara) vectors, respectively. The sgRNA driven by the U6 promoter was incorporated into pUC57. The DNA fragment for the SaCas9 expression cassette was introduced between the inverted terminal repeats of the pAAV plasmid. The AAV genes were packaged by triple plasmid transfection of AAVpro293T cells to produce the AAV vector (helper-free system), as described previously 48 . The titers of recombinant AAV vectors were determined by quantitative PCR, as previously described 49 . Cells (5×10 4 cells/well) were seeded in 48 well plates coated with collagen type I (Cellmatrix type I-C, Nitta Gelatin) the day before transduction. The plasmids (200 ng) were incubated together with Lipofectamine 3000 (Thermo Fisher Scientific), and then directly added to the cell culture according to the manufacturer's recommendations. To obtain stable expressing clones, 400 µg/mL G418 (Nacalai Tesque) was added to the culture medium after the transfection with pBApo-EF1α Neo. The DNAs were isolated at 72 hr after the transduction. DNA fragments at the target site were amplified with ExTaq DNA polymerase (Takara Bio). Purified PCR products were denatured and re-annealed using a thermal cycler, and then treated with T7 endonuclease (Nippon Gene). DNA fragments were analyzed by a MultiNA microchip electrophoresis system. When indicated, PCR amplicons were subjected to 300-bp paired-end read sequencing at the NGS core facility at the Research Institute for Microbial Diseases of Osaka University (Osaka, Japan). The mutation frequencies were assessed by CRISPResso2 45 . Animal experiments All animal experimental procedures were approved by The Institutional Animal Care and Concern Committee of Jichi Medical University (permission number: 20051-10), and animal care was conducted in accordance with the committee’s guidelines and ARRIVE guidelines 50 , 51 . C57BL/6 mice were purchased from SLC Japan (Shizuoka, Japan). The AAV vector was administered intravenously through the jugular vein (100–150 µL) of mice anesthetized with isoflurane (1–3%). To obtain plasma samples, blood samples were drawn from the jugular vein using a 29G micro-syringe (TERUMO) containing 1/10 (volume/volume) sodium citrate. Platelet-poor plasma was obtained by centrifugation and then frozen and stored at − 80°C until analysis. Plasma FIX activity (FIX:C) was measured by a one-stage clotting-time assay with an automated coagulation analyzer (Sysmex CS-1600). Cryo-EM sample preparation and data collection The eSaCas9-NNG–sgRNA–target DNA ternary complex was reconstituted by mixing the purified eSaCas9-NNG, the 98-nt sgRNA, the 43-nt target DNA, and the 43-nt non-target DNA at a molar ratio of 1:1.2:1.25:1.25 at room temperature for 10 min. Each DNA strand contained phosphorothioate modifications within the phosphate backbone around the cleavage site to prevent DNA cleavage (Supplementary Table S3). The SaCas9 and eSaCas9-NNG in complex with the sgRNA and target dsDNA containing the single-nucleotide mismatch at position 21 were reconstituted in the same way, except that the incubation was performed at room temperature for 10 min. The ternary complexes were purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare), equilibrated with buffer E (20 mM HEPES-NaOH, pH 7.6, 50 mM NaCl, 2 mM MgCl 2 , 10 µM ZnCl 2 , and 1 mM DTT). The purified complex solution (A 260 nm = 26) was mixed with 0.005% Tween 20, and then applied to Au 300-mesh R1.2/1.3 grids (Quantifoil) that were glow-discharged in a Vitrobot Mark IV (FEI) at 4°C, with a waiting time of 10 sec and a blotting time of 4 sec under 100% humidity conditions. The grids were plunge-frozen in liquid ethane and cooled to the temperature of liquid nitrogen. Micrographs for all datasets were collected with a Titan Krios G3i microscope (Thermo Fisher Scientific) running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode (The University of Tokyo, Japan). Datasets were collected with a total dose of approximately 50 electrons per Å 2 per 48 frames by the standard mode, using the EPU software (Thermo Fisher Scientific). The dose-fractionated movies were subjected to beam-induced motion correction and dose weighting using Patch Motion Correction, and the contrast transfer function (CTF) parameters were estimated using Patch-based CTF estimation in cryoSPARC v4.4.0 52,53 . Cryo-EM data processing Data were processed with the cryoSPARC v4.4.0 software platform 52 . For the eSaCas9-NNG–sgRNA–target DNA ternary complex, 4,900,536 particles were automatically picked using Template Picker from the 8,625 motion-corrected and dose-weighted micrographs, followed by several rounds of reference-free 2D classification to curate particle sets. Using maps derived from ab-initio reconstruction as templates, 1,659,239 selected particles were subjected to heterogeneous refinement, resulting in the reconstructions of six distinct conformational states. Four of these classes, corresponding to State I (catalytically active state), State II (interrogation state), State III (intermediate state), and State IV (translocation state), were subjected to further processing. For States I, III, and IV, the particles were subjected to CTF refinement, Reference-Based Motion Correction (RBMC), and 3D classification without alignment. Non-uniform refinement after subsequent postprocessing yielded maps at overall resolutions of 3.14 Å (catalytically active state), 2.90 Å (intermediate state), and 2.76 Å (translocation state), according to the Fourier shell correlation (FSC) criterion of 0.143 54,55 . For the interrogation state, the selected particles were subjected to 3D variability analysis 56 . The resulting maps with different conformations were used for subsequent heterogeneous refinement. The particles with the most detailed features after heterogeneous refinement were refined using non-uniform refinement after CTF refinement and RBMC, and yielded a map at an overall resolution of 3.17 Å according to the FSC criterion of 0.143. The local resolution was estimated by BlocRes in cryoSPARC. The datasets for SaCas9 and eSaCas9-NNG in complex with the sgRNA and the mismatched target dsDNA were processed using cryoSPARC in a similar manner as described above. For data processing details, see Extended Data Figs. 4 and 6 . Model building and validation The models were built using the crystal structure of SaCas9 (PDB: 5CZZ) as the reference 9 , followed by manual model building with Coot 57 , 58 . The models were refined using Servalcat against unsharpened half maps 59 . The reference structure restraints were used for the refinement of the catalytically active, interrogation, and intermediate states, which were generated from the AlphaFold2 predicted models and the intermediate and translocation state models using ProSmart 60 , 61 . The stereochemical restraints for phosphorothioate modified DNA links were generated using AceDRG 62 . The models were validated using MolProbity 63 . Molecular graphics figures were prepared with UCSF ChimeraX-1.7.1 64 . QUANTIFICATION AND STATISTICAL ANALYSIS No statistical methods were used to predetermine sample size. Sample size was based on experimental feasibility and sample availability. Samples were processed in random order. Statistical analyses were performed using GraphPad Prism 10 (Graph Pad Software, San Diego, CA). All data are presented as the mean ± standard deviation (s.d.). Declarations Acknowledgements We thank Sachiyo Kamimura and Yuiko Ogihara of Jichi Medical University and Keiko Ogomori of the University of Tokyo for their technical assistance. T.O. was supported by AMED Grant Numbers JP23fk0410037, JP24fk0410061, JP24bm1323001, and JP23ae0201007. H.N. is supported by JSPS KAKENHI Grant Numbers 21H05281 and 22H00403, the Takeda Medical Research Foundation, the Inamori Research Institute for Science, and JST, CREST Grant Number JPMJCR23B6. O.N. was supported by AMED Grant Numbers JP23fa627001 and JP19am0401005, the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED, under grant numbers JP23ama121002 (support number 3272, M.K.) and JP23ama121012 (support no. 4894, O.N.), and the Cabinet Office, Government of Japan, Public/Private R&D Investment Strategic Expansion Program (PRISM) Grant Number JPJ008000. Author contributions S.N.O., R.N., S.K., and H.N. performed biochemical experiments with assistance from S.O. and K.H.; S.I., H.M., S.N.O. and N.Y. performed cell biological experiments with assistance from M.T. and K.H.; Y.K., T.H., and T.O. conducted AAV preparation and mouse experiments; K.J. and S.Q.T. conducted GUIDE-seq analysis; S.N.O. and R.N. performed structural analyses with assistance from H.H., K.Y., Y.I., and H.N.; S.N.O., R.N., H.N., and O.N. wrote the manuscript with help from all authors; H.N. and O.N. supervised the research. Declaration of interests A patent application has been filed related to this work. O.N. is a co-founder, board member, and scientific advisor of Curreio. The remaining authors declare no competing interests. Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Osamu Nureki ( [email protected] ). Data and code availability The atomic models of eSaCas9-NNG–guide RNA–target DNA complexes have been deposited in the Protein Data Bank under the accession codes 8ZCY (interrogation state), 8ZCZ (intermediate state), 8ZD0 (translocation state), and 8ZDA (active state). The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-39941 (interrogation state), EMD-39942 (intermediate state), EMD-39944 (translocation state), and EMD-39954 (active state). The atomic models of SaCas9 and eSaCas9-NNG bound to the sgRNAs and target DNA containing a mismatch at position 21 have been deposited in the Protein Data Bank under the accession codes 9MB6 (SaCas9) and 9MB7 (eSaCas9-NNG). The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-63767 (SaCas9) and EMD-63768 (eSaCas9-NNG). The NGS data have been deposited in the NCBI under accession code PRJNA1088532. 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Supplementary Files SaCas9SupplementaryTable.pdf Supplementary Table SaCas9NNGExtendedFig.pdf Extended Data Fig. 1 | Engineering of the SaCas9-NNG variant. (a) In vitro DNA cleavage activities of SaCas9 with 20–23-nt guide sgRNAs. The linearized plasmid target bearing the TTGAAT PAM was incubated with the SaCas9–sgRNA complex at 37°C for 0.5, 1, 2, and 5 min. The cleavage products were then analyzed by a MultiNA microchip electrophoresis system. (b) Crystal structure of SaCas9 in complex with its sgRNA and target DNA (PDB: 5CZZ). (c) PAM recognition by SaCas9. dG3* is recognized by Arg1015, while dA4*/dA5* and dT6* are recognized by Asn985 and Arg991, respectively. (d) In vitro cleavage activities of SaCas9, AAKRKRS, and AAKRKNRFS toward the target DNA bearing the TTGAAT PAM. (e and f) Structural modeling of the T927K/K929N (e) and I1017F (f) mutations. The mutated residues were manually modeled using the Coot software. (g) In vitro DNA cleavage activities of AAKRKNRFS toward DNA targets with different PAMs. The linearized plasmid targets were incubated with the SaCas9–sgRNA complex at 37°C for 0.5 and 2 min. Data are mean ± s.d. (n = 3). (h) Potential steric clash between the fifth T (dT5*) and Asn986. The side chain of Asn986 clashes with the methyl group of dT5*, resulting in the weak activity of AAKRKNRFS toward the TTGGTT target. dT5* was manually modeled using the Coot software. Extended Data Fig. 2 | Genome- and base-editing activities in mammalian cells. (a) Indel efficiencies of SaCas9 (white) and SaCas9-NNG (orange) at nine endogenous target loci in murine immortalized liver (TLR3) cells. (b and c) A-to-G conversions in HEK293 cells by SaCas9-ABE8e (b) and SaCas9-NNG-ABE8e (c) at various point mutations known to cause hemophilia B. The A-to-G base conversions were mainly observed between the 7th to 20th positions from the PAM within the protospacer. Extended Data Fig. 3 | Engineering of the eSaCas9-NNG variant. (a) Close-up view of the PAM-distal end of the heteroduplex and REC2 domain in the SaCas9 structure (PDB: 5CZZ). Asn413 interacts with the dC19 ribose moiety in the target DNA, while Arg420 forms salt bridges with Glu406 and Asp412 within the REC2 domain. (b) In vitro DNA cleavage kinetics of SaCas9, eSaCas9, SaCas9-NNG, and eSaCas9-NNG. The linearized plasmid target bearing the TTGAAT PAM was incubated with the SaCas9–sgRNA complexes at 37°C for 0.5, 1, 2, and 5 min. The cleavage products were then analyzed by a MultiNA microchip electrophoresis system. (c) Off-target cleavage at the VEGFA site, identified by GUIDE-seq. The on-target sequence is shown in the top line, with detected cleaved sites shown underneath. The read counts are shown to the right of each site. (d) Summarized on- and off-target activities for GUIDE-seq2 results from (c) plotted as a scatter plot. Off-target activities are calculated as the ratio between read counts for the off-target and the corresponding on-target read count. Extended Data Fig. 4 | Cryo-EM analysis of the eSaCas9-NNG–guide RNA–target DNA complex. (a) Size-exclusion chromatography profile of the eSaCas9-NNG–guide RNA–target DNA complex. The peak fraction indicated as a complex was used for cryo-EM analysis. (b) Representative cryo-EM micrograph, recorded on a 300 kV Titan Krios electron microscope with a K3 camera. (c) Representative 2D class average images. (d) Single-particle cryo-EM image processing workflow. Cryo-EM density maps according to the local resolution are shown at the bottom. Data were processed using the cryoSPARC v4.4.0 software platform. (e) Fourier shell correlation curves for the 3D reconstructions. (f) Fourier shell correlation curves calculated between the refined models and the cryo-EM density maps. Extended Data Fig. 5 | Cryo-EM structure of the eSaCas9-NNG–guide RNA–target DNA complex. (a) Domain structure of eSaCas9-NNG. I, RuvC-I; II, RuvC-II; BH, bridge helix; PLL, phosphate lock loop. (b and c) Cryo-EM density maps (b) and ribbon models (c) of the eSaCas9-NNG–guide RNA–target DNA complex. TS, target DNA strand; NTS, non-target DNA strand. (d) Schematic of the sgRNA and the target DNA. The cleavage site in the TS is indicated by a magenta triangle. The disordered regions are enclosed by dashed boxes. Phosphorothioate-modified DNA nucleotides (S-modification) are colored green. (e) Structure of the sgRNA and target DNA complex. The cleavage site in the TS is indicated by a magenta triangle. Extended Data Fig. 6 | Structural comparison between SaCas9 and eSaCas9-NNG. (a) Crystal structure of the SaCas9–guide RNA–target DNA complex (PDB: 5CZZ). (b) Cryo-EM structure of the eSaCas9-NNG–guide RNA–target DNA complex. (c) Locations of the RuvC and HNH domains of eSaCas9-NNG in the active state. (d and e) Close-up views of the HNH (d) and RuvC (e) active sites in eSaCas9-NNG. Cryo-EM density maps are shown as blue meshes. The bound Mg 2+ ions and water molecules are depicted as gray and red spheres, respectively. (f) Cryo-EM density map of the eSaCas9-NNG–guide RNA–target DNA complex around the PAM-containing DNA duplex. Mutated residues are highlighted in red. Cryo-EM density maps are shown as blue meshes. (g) Structural comparison of the PAM recognitions by SaCas9 (light blue) and eSaCas9-NNG (colored as in b). The fourth G in the PAM-duplex (dG4*) was manually modeled using the Coot software. In both structures, Arg1015 forms bidentate hydrogen bonds with the third G in the PAM duplex (dG3*). In the eSaCas9-NNG structure, in contrast, Phe1017 induces a slight displacement of Arg1015, preventing it from stabilizing dG4* through hydrogen-bonding interactions. Extended Data Fig. 7 | Cryo-EM analysis of SaCas9 and eSaCas9-NNG bound to their cognate sgRNA and the mismatched target DNA. (a and b) Size-exclusion chromatography profiles of the SaCas9–guide RNA–mismatched target DNA (a) and eSaCas9-NNG–guide RNA–mismatched target DNA (b) complexes. The peak fractions indicated as a complex were used for cryo-EM analysis. (c and d) Representative cryo-EM micrographs of the SaCas9–guide RNA–mismatched target DNA (c) and eSaCas9-NNG–guide RNA–mismatched target DNA (d) complexes, recorded on a 300 kV Titan Krios electron microscope with a K3 camera. (e and f) Representative 2D class average images of the SaCas9–guide RNA–mismatched target DNA (e) and eSaCas9-NNG–guide RNA–mismatched target DNA (f) complexes. (g and h) Single-particle cryo-EM image processing workflows of the SaCas9–guide RNA–mismatched target DNA (g) and eSaCas9-NNG–guide RNA–mismatched target DNA (h) complexes. Cryo-EM density maps according to the local resolution are shown at the bottom. Data were processed using the cryoSPARC v4.4.0 software platform. (i) Fourier shell correlation curves for the 3D reconstructions. (j) Fourier shell correlation curves calculated between the refined models and the cryo-EM density maps. Extended Data Fig. 8 | Representative cryo-EM densities. (a) Cryo-EM density map of the SaCas9–guide RNA–mismatched target DNA complex around the G1:dG21 mismatch at the PAM-distal end. Cryo-EM density maps are shown as blue meshes. (b) Cryo-EM density map of the eSaCas9-NNG–guide RNA–mismatched target DNA complex around the G1:dG21 mismatch at the PAM-distal end. Cryo-EM density maps are shown as blue meshes. (c) Cryo-EM density map of the SaCas9–guide RNA–mismatched target DNA complex around the REC2 domain and PAM-distal guide–target heteroduplex. Cryo-EM density maps are shown as blue meshes. (d) Cryo-EM density map of the eSaCas9-NNG–guide RNA–mismatched target DNA complex around the REC2 domain and PAM-distal guide–target heteroduplex. Mutated residues are highlighted in red. Cryo-EM density maps are shown as blue meshes. Extended Data Fig. 9 | Conformational changes in eSaCas9-NNG coupled with the guide–target heteroduplex formation. (a and b) Structures of the eSaCas9-NNG–guide RNA–target DNA complexes in the intermediate (a) and translocation (b) states. (c) Superimposition of the intermediate (colored as in a) and translocation (light blue) states. (d and e) Close-up views around the L2 linker and the RuvC domain in the intermediate (d) and translocation (e) states. In the intermediate state, residues 635–645 adopt an α helix structure and interact with both the RuvC and HNH domains, whereas in the translocation state, the L2 linker is melted and the HNH domain is disordered. Hydrogen bonds are shown as dashed lines. (f) Structural comparison of the PAM-distal region between the intermediate and translocation states. The two structures are superimposed based on their RuvC domains. The PAM-distal heteroduplex in the translocation state clashes with the L2 linker in the intermediate state. (g and h) Structures of the eSaCas9-NNG–guide RNA–target DNA complexes in the intermediate (g) and active (h) states. The HNH domains are highlighted in magenta. During the transition from the intermediate state to the active state, the HNH domain undergoes an approximately 140° rotation, docking onto the TS cleavage site. Upon docking of the HNH domain, residues 126–146 in the REC1 domain become disordered due to steric clashes with the HNH domain. (i and j) Surface models of the eSaCas9-NNG–guide RNA–target DNA complexes in the intermediate (i) and active (j) states. The L1 and L2 linkers are highlighted in light green. During the transition from the intermediate state to the active state, the L1 linker undergoes an approximately 180° rotation and binds to the minor groove of the PAM-distal heteroduplex, while the L2 linker adopts a loop conformation and forms an NTS-binding pathway toward the RuvC active site in the active state. (k) Superimposition of the RuvC domains in the intermediate (light blue) and active (colored as in a) states. The catalytic residues in the RuvC active site and the bound Mg 2+ ions in the active state are shown as sticks and gray spheres, respectively. In the active state, the C-terminal region of the β4 strand is melted. (l) Recognition of the dA(−1)-dT1* base pair and the displaced non-target DNA strand. Extended Data Fig. 10 | Conformational changes in SpCas9 and eSaCas9-NNG. (a) Structure of the SpCas9–guide RNA–target DNA complex in the inactive state (PDB: 6O0Z). The HNH domain is distant from the TS cleavage site. The disordered L1 linker is indicated by a light green circle. The TS cleavage site is indicated by a magenta triangle. (b) Structure of the eSaCas9-NNG–guide RNA–target DNA complex in the intermediate state. The HNH domain is distant from the TS cleavage site, and the RuvC active site is occluded by the L1 linker. The TS cleavage site is indicated by a magenta triangle. (c) Conformational change in the HNH domain of SpCas9 during the transition from the inactive state (PDB: 6O0Z) to the active state (PDB: 6O0Y). The HNH domain in the inactive state is superimposed onto the overall structure in the active state. (d) Conformational change in the HNH domain of eSaCas9-NNG during the transition from the intermediate state to the active state. The HNH domain in the intermediate state is superimposed onto the structure in the active state. (e and f) NTS-loading paths toward the RuvC active site in SpCas9 (PDB: 6O0Y) (e) and eSaCas9-NNG (f). (g and h) Recognition of the PAM-distal region of the guide–target heteroduplex by SpCas9 (PDB: 6O0Y) (g) and eSaCas9-NNG (h). SupplementarymovieS1.mp4 Supplementary Movie S1 | Conformational change in the mismatch-sensing helix. Structural comparison between the SaCas9–guide RNA–mismatched target DNA and eSaCas9-NNG–guide RNA–mismatched target DNA complexes reveals the local conformational changes of the mismatch-sensing helix in the REC2 domain. SupplementarymovieS2.mp4 Supplementary Movie S2 | Action mechanism of SaCas9. Structural comparisons between the interrogation, intermediate, translocation and active states of eSaCas9-NNG reveal stepwise domain rearrangements coupled to guide RNA–target DNA heteroduplex formation. SupplementarymovieS3.mp4 Supplementary Movie S3 | Conformational changes in the HNH domain and L1/L2 linker. Structural comparison between the intermediate and active states of eSaCas9-NNG reveals substantial conformational changes in the HNH domain and L1/L2 linker. Cite Share Download PDF Status: Published Journal Publication published 16 Apr, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":106524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEngineering of the SaCas9-NNG variant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of SaCas9 with the 20–23-nt guide sgRNAs. The linearized plasmid target bearing the TTGAAT PAM was incubated with the SaCas9–sgRNA complex at 37°C for 0.5 and 2 min. The cleavage products were then analyzed by a MultiNA microchip electrophoresis system. Data are mean ± s.d. (n = 3).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e)\u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of the N985A/R991A, N985A/R991A/E782K, N985A/R991A/L800R, N985A/R991A/T927K, N985A/R991A/N968R, N985A/R991A/A1021S, and N985A/R991A/E782K/L800R/T927K/N968R/A1021S (AAKRKRS) mutants. A linearized plasmid target bearing the TTGAAT PAM was incubated with the SaCas9–sgRNA complex (50 nM) at 37°C for 2 and 5 min, and the reaction products were then analyzed using a MultiNA microchip electrophoresis system.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec \u003c/strong\u003eand\u003cstrong\u003e d\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of SaCas9 (\u003cstrong\u003ec\u003c/strong\u003e) and SaCas9-NNG (\u003cstrong\u003ed\u003c/strong\u003e) toward DNA targets with different PAMs. The linearized plasmid targets were incubated with the SaCas9–sgRNA complex at 37°C for 0.5 and 2 min. Data are mean ± s.d. (n = 3).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Sequence logos of the PAMs for SaCas9 (left) and SaCas9-NNG (right), obtained from the PAM identification assay.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of SaCas9, SaCas9-NNG, and SaCas9-KKH toward DNA targets with four different PAMs. The linearized plasmid targets were incubated with the SaCas9–sgRNA complex at 37°C for 0.5 and 2 min. Data are mean ± s.d. (n = 3).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/215e6bf8cc554ee29a69f588.png"},{"id":91821661,"identity":"f433d4ea-1395-4e5b-8b5a-e30dbc483e51","added_by":"auto","created_at":"2025-09-22 07:30:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome- and base-editing activities in human cells and mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea \u003c/strong\u003eand\u003cstrong\u003e b\u003c/strong\u003e) Efficiencies of indel formation (\u003cstrong\u003ea\u003c/strong\u003e) and C-to-T conversions (\u003cstrong\u003eb\u003c/strong\u003e) by SaCas9 (gray) and SaCas9-NNG (orange) at endogenous target sites in HEK293Ta cells. Data are mean ± s.d. (n = 3)\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) A schematic of the AAV vector used for mouse liver genome editing.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Indel frequency in mouse livers. Data are mean ± s.d. (n = 4). Statistical significance between SaCas9 and SaCas9-NNG was analyzed by two-tailed Student’s t test.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Time course of plasma factor IX activities (FIX:C) in mouse livers.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/e24b16dafb637ee2ff9bda73.png"},{"id":91820989,"identity":"c4298e04-ccf5-4da2-b3b3-a56848fb00d8","added_by":"auto","created_at":"2025-09-22 07:22:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":145390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEngineering of the eSaCas9 variant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of SaCas9, N413A, R420A, eSaCas9 (N413A/R420A), SaCas9-NNG, eSaCas9-NNG, and SaCas9-HF\u003csup\u003e30\u003c/sup\u003e toward a fully matched on-target DNA and an off-target DNA with a mismatch at position 21 from the PAM. The linearized plasmid targets were incubated with the SaCas9–sgRNA complex at 37°C for 0.5 and 2 min. Data are mean ± s.d. (n = 3).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e)\u003cem\u003e In vitro\u003c/em\u003e DNA cleavage activities of SaCas9, eSaCas9, SaCas9-NNG, eSaCas9-NNG, and SaCas9-HF toward a fully matched on-target DNA and off-target DNAs containing a mismatch at positions 1–21. The linearized plasmid targets were incubated with the SaCas9–sgRNA complex at 37°C for 3 min. Data are mean ± s.d. (n = 3).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e)\u003cem\u003e \u003c/em\u003eIndel formation efficiencies of SaCas9 (white), eSaCas9 (gray), SaCas9-NNG (orange), and eSaCas9-NNG (red) at endogenous target sites in HEK293Ta cells. Data are mean ± s.d. (n = 3).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Schematic representation of the endogenous target loci selected for comparing the indel efficiencies of eSaCas9-NNG, SpRY, SpG, and iGeoCas9. Three target sites with NGGNC PAMs (above) were chosen, which are accessible by eSaCas9-NNG, SpG (NG PAM), SpRY (virtually no PAM restrictions), and iGeoCas9 (NNNNC PAM). Additionally, three target sites with NTGNW PAMs were selected (below), which can only be targeted by eSaCas9-NNG or SpRY. While 21-nt guide sgRNAs were used for eSaCas9-NNG and iGeoCas9, 20-nt guide sgRNAs were used for SpRY and SpG for their optimal activities.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e)\u003cem\u003e \u003c/em\u003eIndel formation efficiencies of eSaCas9-NNG (pink), SpRY (purple), SpG (blue), and iGeoCas9 (red) at endogenous target sites in HEK293T cells. Data are mean ± s.d. (n = 3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/9ddfbc85a63cdabf1121ddb8.png"},{"id":91821662,"identity":"81669839-8e8f-49b7-9a87-ed906942e27d","added_by":"auto","created_at":"2025-09-22 07:30:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":557028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural basis for relaxed PAM recognition and improved specificity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Recognition of the relaxed NNG PAM. Mutated residues are highlighted in red. Hydrogen bonds are shown as dashed lines.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ec\u003c/strong\u003e) Structures of SaCas9 (\u003cstrong\u003eb\u003c/strong\u003e) and eSaCas9-NNG (\u003cstrong\u003ec\u003c/strong\u003e) bound to the sgRNAs and target DNA containing a G:dG mismatch at position 21 from the PAM.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Structural comparison between SaCas9 (light blue) and eSaCas9-NNG (colored as in \u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e) Close-up views around the REC2 domain and the PAM-distal region of the guide–target heteroduplex in SaCas9 (\u003cstrong\u003ee\u003c/strong\u003e) and eSaCas9-NNG (\u003cstrong\u003ef\u003c/strong\u003e). The G1:dG21 mismatch at the PAM-distal end forms a Hoogsteen base pair in both SaCas9 and eSaCas9-NNG. Hydrogen bonds are shown as dashed lines.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg\u003c/strong\u003e) Structural comparison of the PAM-distal region between SaCas9 and eSaCas9-NNG. In eSaCas9-NNG, the MSH undergoes an outward displacement from the PAM-distal guide–target heteroduplex due to the N413A/R420A mutations, and Asn419 adopts a flipped-out conformation.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/7ecff997879da9a85ab580e7.png"},{"id":91820994,"identity":"c85f8055-29a4-4a1e-a301-18c6729a634c","added_by":"auto","created_at":"2025-09-22 07:22:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":710062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structures of eSaCas9-NNG in distinct functional states.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e–\u003cstrong\u003ec\u003c/strong\u003e) Cryo-EM density map (top) and structural models of the entire complex (middle) and the nucleic acids (bottom) of the eSaCas9-NNG–guide RNA–target DNA complexes in the interrogation (\u003cstrong\u003ea\u003c/strong\u003e), intermediate (\u003cstrong\u003eb\u003c/strong\u003e), and translocation (\u003cstrong\u003ec\u003c/strong\u003e) states.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Recognition of the PAM in the interrogation state.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Recognition of the PAM-proximal region of the guide RNA–target DNA heteroduplex in the intermediate state.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Close-up view of the RuvC active site in the translocation state. The magnesium ion coordinated with Asp10 and His701 is shown as a gray sphere. In (\u003cstrong\u003ed\u003c/strong\u003e)–(\u003cstrong\u003ef\u003c/strong\u003e), cryo-EM density maps are shown as blue meshes.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/5fb9066b60b01d21b01d3f90.png"},{"id":91820967,"identity":"ec2622b4-67a2-412f-aa01-1997ab986977","added_by":"auto","created_at":"2025-09-22 07:22:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":455661,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation mechanisms of SpCas9 and SaCas9.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematics showing conformational changes in SpCas9 (top) and SaCas9 (bottom) during DNA cleavage. First, both SpCas9 and SaCas9 bind to a linear target dsDNA to interrogate their cognate PAM sequences. In SpCas9, the REC1 and REC2 domains are ordered and adopt an open conformation (PDB: 7S3H), while in SaCas9, the entire REC lobe is disordered. The dsDNA is then unwound and the guide–target heteroduplex is formed. In SpCas9, the entire REC lobe is ordered when a 6-bp guide–target heteroduplex forms, creating a positively charged cleft that accommodates the PAM-distal target DNA duplex (PDB: 7Z4C). By contrast, in SaCas9, even when an 18-bp guide–target heteroduplex forms, the REC1 domain is ordered, while the REC2 domain remains disordered. Subsequently, in SpCas9, the formation of a 12-bp heteroduplex induces structural disorder in the HNH domain (PDB: 7Z4G), whereas the formation of an 18-bp heteroduplex in SaCas9 does not induce such conformational changes. In SaCas9, the formation of the 21-bp heteroduplex causes the disordering of the HNH domain. Finally, the HNH domains of SpCas9 and SaCas9 commonly undergo significant rotations, docking onto the TS cleavage site, with the L2 linker forming loading paths for the NTS toward the RuvC active sites. Upon HNH domain docking, the REC2 domain of SpCas9 becomes disordered (PDB: 6O0Y)\u003csup\u003e38\u003c/sup\u003e or moves outward (PDBs: 7S4X and 7Z4J)\u003csup\u003e31,33\u003c/sup\u003e, while residues 126–146 in the REC1 domain become disordered in eSaCas9-NNG. The RuvC and HNH domains are indicated with yellow stars.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/070f59e0109d9a299761649a.png"},{"id":107206885,"identity":"00870cb6-2d3f-4506-afc5-4cc53054e367","added_by":"auto","created_at":"2026-04-18 07:05:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2498912,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/a60a1435-689f-4690-a261-f8a28d454e8f.pdf"},{"id":91820980,"identity":"7e1ddd64-d4af-4de0-ba71-fe31e48ebed9","added_by":"auto","created_at":"2025-09-22 07:22:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":202615,"visible":true,"origin":"","legend":"Supplementary Table","description":"","filename":"SaCas9SupplementaryTable.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/d73fde09147b804da507d6d5.pdf"},{"id":91820984,"identity":"a8279572-3c38-49be-b64b-1f3bbdc0b6c1","added_by":"auto","created_at":"2025-09-22 07:22:47","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17983771,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 1 | Engineering of the SaCas9-NNG variant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of SaCas9 with 20–23-nt guide sgRNAs. The linearized plasmid target bearing the TTGAAT PAM was incubated with the SaCas9–sgRNA complex at 37°C for 0.5, 1, 2, and 5 min. The cleavage products were then analyzed by a MultiNA microchip electrophoresis system.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Crystal structure of SaCas9 in complex with its sgRNA and target DNA (PDB: 5CZZ).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) PAM recognition by SaCas9. dG3* is recognized by Arg1015, while dA4*/dA5* and dT6* are recognized by Asn985 and Arg991, respectively.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e)\u003cem\u003e In vitro \u003c/em\u003ecleavage activities of SaCas9, AAKRKRS, and AAKRKNRFS toward the target DNA bearing the TTGAAT PAM.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e) Structural modeling of the T927K/K929N (\u003cstrong\u003ee\u003c/strong\u003e) and I1017F (\u003cstrong\u003ef\u003c/strong\u003e) mutations. The mutated residues were manually modeled using the Coot software.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of AAKRKNRFS toward DNA targets with different PAMs. The linearized plasmid targets were incubated with the SaCas9–sgRNA complex at 37°C for 0.5 and 2 min. Data are mean ± s.d. (n = 3).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eh\u003c/strong\u003e) Potential steric clash between the fifth T (dT5*) and Asn986. The side chain of Asn986 clashes with the methyl group of dT5*, resulting in the weak activity of AAKRKNRFS toward the TTGGTT target. dT5* was manually modeled using the Coot software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 2 | Genome- and base-editing activities in mammalian cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eIndel efficiencies of SaCas9 (white) and SaCas9-NNG (orange) at nine endogenous target loci in murine immortalized liver (TLR3) cells.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb \u003c/strong\u003eand\u003cstrong\u003e c\u003c/strong\u003e) A-to-G conversions in HEK293 cells by SaCas9-ABE8e (\u003cstrong\u003eb\u003c/strong\u003e) and SaCas9-NNG-ABE8e (\u003cstrong\u003ec\u003c/strong\u003e) at various point mutations known to cause hemophilia B. The A-to-G base conversions were mainly observed between the 7th to 20th positions from the PAM within the protospacer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 3 | Engineering of the eSaCas9-NNG variant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Close-up view of the PAM-distal end of the heteroduplex and REC2 domain in the SaCas9 structure (PDB: 5CZZ). Asn413 interacts with the dC19 ribose moiety in the target DNA, while Arg420 forms salt bridges with Glu406 and Asp412 within the REC2 domain.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage kinetics of SaCas9, eSaCas9, SaCas9-NNG, and eSaCas9-NNG. The linearized plasmid target bearing the TTGAAT PAM was incubated with the SaCas9–sgRNA complexes at 37°C for 0.5, 1, 2, and 5 min. The cleavage products were then analyzed by a MultiNA microchip electrophoresis system.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Off-target cleavage at the \u003cem\u003eVEGFA\u003c/em\u003e site, identified by GUIDE-seq. The on-target sequence is shown in the top line, with detected cleaved sites shown underneath. The read counts are shown to the right of each site.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Summarized on- and off-target activities for GUIDE-seq2 results from (\u003cstrong\u003ec\u003c/strong\u003e) plotted as a scatter plot. Off-target activities are calculated as the ratio between read counts for the off-target and the corresponding on-target read count.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 4 | Cryo-EM analysis of the eSaCas9-NNG–guide RNA–target DNA complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Size-exclusion chromatography profile of the eSaCas9-NNG–guide RNA–target DNA complex. The peak fraction indicated as a complex was used for cryo-EM analysis.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Representative cryo-EM micrograph, recorded on a 300 kV Titan Krios electron microscope with a K3 camera.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Representative 2D class average images.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Single-particle cryo-EM image processing workflow. Cryo-EM density maps according to the local resolution are shown at the bottom. Data were processed using the cryoSPARC v4.4.0 software platform.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Fourier shell correlation curves for the 3D reconstructions.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Fourier shell correlation curves calculated between the refined models and the cryo-EM density maps.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 5 | Cryo-EM structure of the eSaCas9-NNG–guide RNA–target DNA complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Domain structure of eSaCas9-NNG. I, RuvC-I; II, RuvC-II; BH, bridge helix; PLL, phosphate lock loop.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ec\u003c/strong\u003e) Cryo-EM density maps (\u003cstrong\u003eb\u003c/strong\u003e) and ribbon models (\u003cstrong\u003ec\u003c/strong\u003e) of the eSaCas9-NNG–guide RNA–target DNA complex. TS, target DNA strand; NTS, non-target DNA strand.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Schematic of the sgRNA and the target DNA. The cleavage site in the TS is indicated by a magenta triangle. The disordered regions are enclosed by dashed boxes. Phosphorothioate-modified DNA nucleotides (S-modification) are colored green.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Structure of the sgRNA and target DNA complex. The cleavage site in the TS is indicated by a magenta triangle.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eExtended Data Fig. 6 | Structural comparison between SaCas9 and eSaCas9-NNG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Crystal structure of the SaCas9–guide RNA–target DNA complex (PDB: 5CZZ).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Cryo-EM structure of the eSaCas9-NNG–guide RNA–target DNA complex.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Locations of the RuvC and HNH domains of eSaCas9-NNG in the active state.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003ee\u003c/strong\u003e) Close-up views of the HNH (\u003cstrong\u003ed\u003c/strong\u003e) and RuvC (\u003cstrong\u003ee\u003c/strong\u003e) active sites in eSaCas9-NNG. Cryo-EM density maps are shown as blue meshes. The bound Mg\u003csup\u003e2+\u003c/sup\u003e ions and water molecules are depicted as gray and red spheres, respectively.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Cryo-EM density map of the eSaCas9-NNG–guide RNA–target DNA complex around the PAM-containing DNA duplex. Mutated residues are highlighted in red. Cryo-EM density maps are shown as blue meshes.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg\u003c/strong\u003e) Structural comparison of the PAM recognitions by SaCas9 (light blue) and eSaCas9-NNG (colored as in \u003cstrong\u003eb\u003c/strong\u003e). The fourth G in the PAM-duplex (dG4*) was manually modeled using the Coot software. In both structures, Arg1015 forms bidentate hydrogen bonds with the third G in the PAM duplex (dG3*). In the eSaCas9-NNG structure, in contrast, Phe1017 induces a slight displacement of Arg1015, preventing it from stabilizing dG4* through hydrogen-bonding interactions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 7 | Cryo-EM analysis of SaCas9 and eSaCas9-NNG bound to their cognate sgRNA and the mismatched target DNA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea \u003c/strong\u003eand\u003cstrong\u003e b\u003c/strong\u003e) Size-exclusion chromatography profiles of the SaCas9–guide RNA–mismatched target DNA (\u003cstrong\u003ea\u003c/strong\u003e) and eSaCas9-NNG–guide RNA–mismatched target DNA (\u003cstrong\u003eb\u003c/strong\u003e) complexes. The peak fractions indicated as a complex were used for cryo-EM analysis.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec \u003c/strong\u003eand\u003cstrong\u003e d\u003c/strong\u003e) Representative cryo-EM micrographs of the SaCas9–guide RNA–mismatched target DNA (\u003cstrong\u003ec\u003c/strong\u003e) and eSaCas9-NNG–guide RNA–mismatched target DNA (\u003cstrong\u003ed\u003c/strong\u003e) complexes, recorded on a 300 kV Titan Krios electron microscope with a K3 camera.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee \u003c/strong\u003eand\u003cstrong\u003e f\u003c/strong\u003e) Representative 2D class average images of the SaCas9–guide RNA–mismatched target DNA (\u003cstrong\u003ee\u003c/strong\u003e) and eSaCas9-NNG–guide RNA–mismatched target DNA (\u003cstrong\u003ef\u003c/strong\u003e) complexes.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg \u003c/strong\u003eand\u003cstrong\u003e h\u003c/strong\u003e) Single-particle cryo-EM image processing workflows of the SaCas9–guide RNA–mismatched target DNA (\u003cstrong\u003eg\u003c/strong\u003e) and eSaCas9-NNG–guide RNA–mismatched target DNA (\u003cstrong\u003eh\u003c/strong\u003e) complexes. Cryo-EM density maps according to the local resolution are shown at the bottom. Data were processed using the cryoSPARC v4.4.0 software platform.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ei\u003c/strong\u003e) Fourier shell correlation curves for the 3D reconstructions.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ej\u003c/strong\u003e) Fourier shell correlation curves calculated between the refined models and the cryo-EM density maps.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 8 | Representative cryo-EM densities.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Cryo-EM density map of the SaCas9–guide RNA–mismatched target DNA complex around the G1:dG21 mismatch at the PAM-distal end. Cryo-EM density maps are shown as blue meshes.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Cryo-EM density map of the eSaCas9-NNG–guide RNA–mismatched target DNA complex around the G1:dG21 mismatch at the PAM-distal end. Cryo-EM density maps are shown as blue meshes.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Cryo-EM density map of the SaCas9–guide RNA–mismatched target DNA complex around the REC2 domain and PAM-distal guide–target heteroduplex. Cryo-EM density maps are shown as blue meshes.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Cryo-EM density map of the eSaCas9-NNG–guide RNA–mismatched target DNA complex around the REC2 domain and PAM-distal guide–target heteroduplex. Mutated residues are highlighted in red. Cryo-EM density maps are shown as blue meshes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 9 | Conformational changes in eSaCas9-NNG coupled with the guide–target heteroduplex formation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003eb\u003c/strong\u003e) Structures of the eSaCas9-NNG–guide RNA–target DNA complexes in the intermediate (\u003cstrong\u003ea\u003c/strong\u003e) and translocation (\u003cstrong\u003eb\u003c/strong\u003e) states.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Superimposition of the intermediate (colored as in \u003cstrong\u003ea\u003c/strong\u003e) and translocation (light blue) states.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003ee\u003c/strong\u003e) Close-up views around the L2 linker and the RuvC domain in the intermediate (\u003cstrong\u003ed\u003c/strong\u003e) and translocation (\u003cstrong\u003ee\u003c/strong\u003e) states. In the intermediate state, residues 635–645 adopt an α helix structure and interact with both the RuvC and HNH domains, whereas in the translocation state, the L2 linker is melted and the HNH domain is disordered. Hydrogen bonds are shown as dashed lines.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Structural comparison of the PAM-distal region between the intermediate and translocation states. The two structures are superimposed based on their RuvC domains. The PAM-distal heteroduplex in the translocation state clashes with the L2 linker in the intermediate state.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e) Structures of the eSaCas9-NNG–guide RNA–target DNA complexes in the intermediate (\u003cstrong\u003eg\u003c/strong\u003e) and active (\u003cstrong\u003eh\u003c/strong\u003e) states. The HNH domains are highlighted in magenta. During the transition from the intermediate state to the active state, the HNH domain undergoes an approximately 140° rotation, docking onto the TS cleavage site. Upon docking of the HNH domain, residues 126–146 in the REC1 domain become disordered due to steric clashes with the HNH domain.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ei \u003c/strong\u003eand \u003cstrong\u003ej\u003c/strong\u003e) Surface models of the eSaCas9-NNG–guide RNA–target DNA complexes in the intermediate (\u003cstrong\u003ei\u003c/strong\u003e) and active (\u003cstrong\u003ej\u003c/strong\u003e) states. The L1 and L2 linkers are highlighted in light green. During the transition from the intermediate state to the active state, the L1 linker undergoes an approximately 180° rotation and binds to the minor groove of the PAM-distal heteroduplex, while the L2 linker adopts a loop conformation and forms an NTS-binding pathway toward the RuvC active site in the active state.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ek\u003c/strong\u003e) Superimposition of the RuvC domains in the intermediate (light blue) and active (colored as in \u003cstrong\u003ea\u003c/strong\u003e) states. The catalytic residues in the RuvC active site and the bound Mg\u003csup\u003e2+ \u003c/sup\u003eions in the active state are shown as sticks and gray spheres, respectively. In the active state, the C-terminal region of the β4 strand is melted.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003el\u003c/strong\u003e) Recognition of the dA(−1)-dT1* base pair and the displaced non-target DNA strand.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 10 | Conformational changes in SpCas9 and eSaCas9-NNG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Structure of the SpCas9–guide RNA–target DNA complex in the inactive state (PDB: 6O0Z). The HNH domain is distant from the TS cleavage site. The disordered L1 linker is indicated by a light green circle. The TS cleavage site is indicated by a magenta triangle.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Structure of the eSaCas9-NNG–guide RNA–target DNA complex in the intermediate state. The HNH domain is distant from the TS cleavage site, and the RuvC active site is occluded by the L1 linker. The TS cleavage site is indicated by a magenta triangle.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Conformational change in the HNH domain of SpCas9 during the transition from the inactive state (PDB: 6O0Z) to the active state (PDB: 6O0Y). The HNH domain in the inactive state is superimposed onto the overall structure in the active state.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Conformational change in the HNH domain of eSaCas9-NNG during the transition from the intermediate state to the active state. The HNH domain in the intermediate state is superimposed onto the structure in the active state.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e) NTS-loading paths toward the RuvC active site in SpCas9 (PDB: 6O0Y) (\u003cstrong\u003ee\u003c/strong\u003e) and eSaCas9-NNG (\u003cstrong\u003ef\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e) Recognition of the PAM-distal region of the guide–target heteroduplex by SpCas9 (PDB: 6O0Y) (\u003cstrong\u003eg\u003c/strong\u003e) and eSaCas9-NNG (\u003cstrong\u003eh\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"SaCas9NNGExtendedFig.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/7263c76cc484d38697e282e4.pdf"},{"id":91820995,"identity":"669ac557-2d70-45a3-80b3-55851f89ff6e","added_by":"auto","created_at":"2025-09-22 07:22:48","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":21692318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Movie S1 | Conformational change in the mismatch-sensing helix.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural comparison between the SaCas9–guide RNA–mismatched target DNA and eSaCas9-NNG–guide RNA–mismatched target DNA complexes reveals the local conformational changes of the mismatch-sensing helix in the REC2 domain.\u003c/p\u003e","description":"","filename":"SupplementarymovieS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/f2328e4b85d1eed2fae9dab3.mp4"},{"id":91820978,"identity":"c7229eab-f64e-4dbc-9968-52a2f8a8066b","added_by":"auto","created_at":"2025-09-22 07:22:46","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":69805396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Movie S2 | Action mechanism of SaCas9.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural comparisons between the interrogation, intermediate, translocation and active states of eSaCas9-NNG reveal stepwise domain rearrangements coupled to guide RNA–target DNA heteroduplex formation.\u003c/p\u003e","description":"","filename":"SupplementarymovieS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/41e512272460325e66e717fd.mp4"},{"id":91820996,"identity":"22179f82-2f0f-4e00-ab0a-7c8f342090a2","added_by":"auto","created_at":"2025-09-22 07:22:48","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":29689599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Movie S3 | Conformational changes in the HNH domain and L1/L2 linker.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural comparison between the intermediate and active states of eSaCas9-NNG reveals substantial conformational changes in the HNH domain and L1/L2 linker.\u003c/p\u003e","description":"","filename":"SupplementarymovieS3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-7614961/v1/cab82829904cf4b7ac0805a7.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Activation mechanism and molecular engineering of Staphylococcus aureus Cas9","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe RNA-guided DNA endonuclease Cas9 associates with a single-guide RNA (sgRNA) to cleave double-stranded DNA targets (dsDNA) complementary to the sgRNA guide. Since \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e Cas9 (SpCas9) exhibits high nuclease activity, it has been widely used for genome editing in eukaryotic cells\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Besides the guide RNA\u0026ndash;target DNA complementarity, SpCas9 requires an NGG (where N is any nucleobase) sequence as the protospacer adjacent motif (PAM), restricting the targetable genomic sites\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. To relax this constraint, we and others have engineered SpCas9 variants with altered PAM specificities, such as SpCas9-NG\u003csup\u003e5\u003c/sup\u003e and SpG/SpRY\u003csup\u003e6\u003c/sup\u003e. Although these SpCas9 variants enable genome editing at expanded target sites in various cell lines, their gene sizes (1,368 residues and 4.1 kb) pose a challenge in packaging them into an adeno-associated virus (AAV) vector for delivery into the target tissue, hampering their therapeutic applications\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e Cas9 (SaCas9) consists of 1,053 residues (3.2 kb), approximately 0.95 kb shorter than SpCas9, and exhibits genome editing activities comparable to those of SpCas9 in human cells\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Notably, SaCas9, along with its sgRNA, can be packaged into a single AAV vector, enabling genome editing in mouse liver\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Furthermore, the catalytically inactive version of SaCas9 (dSaCas9) fused to a transcriptional regulator or the nickase version of SaCas9 (nSaCas9) fused to a cytosine or adenosine deaminase can be utilized as compact tools for transcriptional regulation or base editing, respectively\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, SaCas9 requires relatively long NNGRRT (where R is A or G) PAMs, limiting its utility in genome editing applications\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Furthermore, the crystal structure of SaCas9 represents a catalytically inactive state, wherein the HNH nuclease domain is distant from the cleavage site in the target DNA strand (TS), likely due to the absence of the complementary non-target DNA strand (NTS)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Therefore, the activation mechanism of SaCas9 remains enigmatic.\u003c/p\u003e\u003cp\u003eHere, we rationally engineered a SaCas9 variant (eSaCas9-NNG) that recognizes relaxed NNG PAMs and exhibits reduced off-target cleavage, and demonstrated that eSaCas9-NNG can efficiently edit target sites with NNG PAMs in human cells and mice. We then determined the cryo-electron microscopy (cryo-EM) structures of eSaCas9-NNG in complex with its cognate sgRNA and dsDNA target in five distinct functional states, explaining how the introduced mutations alter the PAM specificity and enhance the cleavage fidelity. Our structures in multiple states reveal the stepwise domain rearrangements coupled to guide RNA\u0026ndash;target DNA heteroduplex formation, highlighting the differences in the activation mechanisms between the small SaCas9 and the large SpCas9. Overall, this study demonstrates that the newly engineered SaCas9 variant can be harnessed as a compact and precise AAV-deliverable genome editing tool, and advances our understanding of the RNA-guided DNA cleavage mechanisms of the diverse Cas9 enzymes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStructure-guided engineering of the SaCas9-NNG variant\u003c/h2\u003e\u003cp\u003eTo determine the optimal guide length for SaCas9, we performed \u003cem\u003ein vitro\u003c/em\u003e cleavage experiments, using the purified SaCas9, sgRNAs with 20- to 23-nt guide segments (sgRNA20\u0026ndash;23), and linearized plasmid DNA containing a target sequence and the canonical TTGAAT PAM (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). SaCas9 with all sgRNAs efficiently cleaved the DNA target, and sgRNA21 showed superior activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), consistent with a previous study showing that 21-nt guide sgRNAs are optimal for SaCas9-mediated genome editing in human cells\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Therefore, we employed sgRNAs with 21-nt guides for the following experiments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo expand the targeting range of SaCas9, we sought to engineer a SaCas9 variant with relaxed recognition for the fourth to sixth positions in the NNGRRT PAM. Our previous structural analysis revealed that the third G in the PAM is recognized by Arg1015 in SaCas9, while the fourth and fifth Rs and the sixth T are recognized by Asn985 and Arg991, respectively\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb,c). We and others previously reported that PAM recognition can be relaxed by combining the elimination of base-specific interactions with PAM nucleotides and the introduction of non-base-specific backbone interactions with the PAM duplex\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Thus, we first purified the SaCas9 N985A/R991A variant, and measured its \u003cem\u003ein vitro\u003c/em\u003e cleavage activity toward a target DNA bearing the TTGAAT PAM. As expected, the N985A/R991A variant showed almost no activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We then examined whether the N985A/R991A activity could be restored by replacing the residues surrounding the PAM duplex with basic or hydrophilic residues, and found that the E782K, L800R, T927K, N968R, and A1021S mutations partially restored the DNA cleavage activity toward the TTGAAT target (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The combination of all these mutations (N985A/R991A/E782K/L800R/T927K/N968R/A1021S; referred to as AAKRKRS) further enhanced the DNA cleavage activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). However, the cleavage rate of AAKRKRS was still slightly lower than that of wild-type SaCas9 (referred to as SaCas9 for simplicity) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Molecular modeling suggested that the K929N and I1017F mutations form hydrogen-bonding and van der Waals interactions with Lys927 (T927K) and Arg1015, respectively, thereby stabilizing the interactions with the PAM duplex (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee,f). Indeed, the inclusion of these two mutations into the AAKRKRS variant enhanced the cleavage activity toward the TTGAAT target to a level comparable to that of SaCas9 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eTo investigate whether the AAKRKNRFS (N985A/R991A/E782K/L800R/T927K/K929N/N968R/I1017F/A1021S) variant exhibits relaxed PAM recognition, we assessed the cleavage activities of SaCas9 and AAKRKNRFS toward target DNAs with 19 different PAMs, including TTG\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eNN\u003c/span\u003eT and TTGAA\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eN\u003c/span\u003e PAMs. SaCas9 efficiently cleaved the target DNAs with TTGRRN PAMs and showed a slight preference for T at the sixth position (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), consistent with the previous study\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In contrast, AAKRKNRFS efficiently cleaved all target DNAs except for the TTGGTT target (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). The SaCas9 structure suggested a steric clash between the side chain of Asn986 and the methyl group of the fifth T, which could reduce the activity of AAKRKNRFS toward the TTGGTT target (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Indeed, the addition of the N986S mutation (N985A/R991A/E782K/L800R/T927K/K929N/N968R/N986S/I1017F/A1021S; referred to as AAKRKNRSFS) enhanced the cleavage activity toward the TTGGTT target, although this variant still showed relatively lower activities for TTGGNN PAMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). To comprehensively explore the PAM specificities of AAKRKNRSFS, we performed \u003cem\u003ein vitro\u003c/em\u003e PAM discovery assays, using a DNA library containing the target sequence adjacent to a randomized 8-bp sequence. We confirmed that, whereas SaCas9 is specific to NNGRR PAMs, AAKRKNRSFS recognizes simple NNG sequences as the PAMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). These results demonstrated that the engineered AAKRKNRSFS variant exhibits a relaxed PAM constraint, and thus we refer to it as SaCas9-NNG.\u003c/p\u003e\u003cp\u003eA previous study reported the SaCas9-KKH variant (E782K/N968K/R1015H), which was engineered via directed evolution and recognizes relaxed NNNRRT PAMs\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. To compare the PAM preferences between SaCas9-NNG and SaCas9-KKH, we evaluated their \u003cem\u003ein vitro\u003c/em\u003e cleavage activities toward target DNAs bearing TT\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eN\u003c/span\u003eAAT PAMs. SaCas9-NNG exhibited no activity except for the TTGAAT target, whereas SaCas9-KKH showed activity against all the PAMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), consistent with the reported relaxed preference of SaCas9-KKH for the third G in the PAM. Nonetheless, under our assay conditions (50 nM Cas9), SaCas9-NNG was much more active than SaCas9-KKH toward the TTGAAT target (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), demonstrating the superiority of SaCas9-NNG for targeting the NNG PAM targets.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenome editing by SaCas9-NNG in mammalian cells\u003c/h3\u003e\n\u003cp\u003eTo assess the genome editing activity of SaCas9-NNG, we measured indel (insertions or deletions) formations induced by SaCas9 and SaCas9-NNG at 35 endogenous target sites with NNG PAMs in human embryonic kidney (HEK) 293T cells (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). As expected, SaCas9 induced indels at the NNGRR, but not NNGYY (where Y is T or C), target sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In contrast, SaCas9-NNG efficiently induced indels at the NNGRR sites (except for the NNGGG sites) and the NNGYY sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), consistent with our \u003cem\u003ein vitro\u003c/em\u003e cleavage data. We also measured the genome-editing efficiencies of SaCas9 and SaCas9-NNG in murine immortalized liver (TLR3) cells. SaCas9 efficiently edited only NNGRR sites, whereas SaCas9-NNG modified all NNG sites, albeit with lower activity at the NNGG sites (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). These results demonstrated that SaCas9-NNG can edit target sites with NNG PAMs in mammalian cells, although with relatively reduced activity at NNGG PAM targets.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eBase editing by SaCas9-NNG in human cells\u003c/h3\u003e\n\u003cp\u003eNext, we examined whether SaCas9-NNG can be harnessed for base-editing technology. We designed the D10A nickase versions of SaCas9 and SaCas9-NNG fused to the activation-induced cytidine deaminase (referred to as SaCas9-AID and SaCas9-NNG-AID, respectively), as in the SpCas9-based cytidine base editor\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and measured C-to-T conversion efficiencies at 35 endogenous target sites with NNG PAMs (identical to those tested for indel formation) in HEK293T cells (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). SaCas9-AID efficiently mediated C-to-T conversions at the NNGRR, but not NNGYY, target sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast, SaCas9-NNG-AID showed C-to-T base conversions toward all the target sites, albeit with lower efficiencies at the NNGG sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Furthermore, we designed the nickase versions of SaCas9 and SaCas9-NNG fused to TadA-8e (referred to as SaCas9-ABE8e and SaCas9-NNG-ABE8e, respectively), as in the SpCas9-based adenine base editor\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and measured A-to-G conversions toward various point mutations known to cause hemophilia B\u003csup\u003e21\u003c/sup\u003e in HEK293 cells (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). SaCas9-ABE8e exhibited A-to-G conversions only at NNGRR targets with relatively low efficiencies, whereas SaCas9-NNG-ABE8e induced A-to-G conversions at all targets (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c). A-to-G base conversions predominantly occurred between the 7th and 20th positions from the PAM within the protospacer (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c), largely consistent with the previously reported target window of SaCas9-ABEmax\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These results demonstrated that the catalytically inactive version of SaCas9-NNG can serve as a useful RNA-guided DNA-targeting platform.\u003c/p\u003e\n\u003ch3\u003eGenome editing by SaCas9-NNG in mice\u003c/h3\u003e\n\u003cp\u003eSince SaCas9 (1,053 residues) is smaller than SpCas9 (1,368 residues), the SaCas9-NNG gene, along with its sgRNA and/or accessory components, can be packaged into an all-in-one AAV vector, enabling its genome-editing applications in living organisms. We designed a single AAV vector, encoding SaCas9 or SaCas9-NNG under the HCRhAAT promoter and a U6 promoter-driven sgRNA targeting the \u003cem\u003eF9\u003c/em\u003e gene for hemophilia B (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). We injected 7-week-old mice with 1\u0026times;10\u003csup\u003e12\u003c/sup\u003e vector genomes (vg) of the single AAV serotype 8 vector, and measured the indel formation at 12 weeks after the injection. SaCas9 induced indels at the NNGGAT and NNGAAA, but not NNGCAA and NNGTCA, target sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In contrast, SaCas9-NNG efficiently induced indels at all the targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Consistently, SaCas9-mediated editing of the \u003cem\u003eF9\u003c/em\u003e target with the NNGGAT and NNGAAA PAMs reduced the plasma coagulation factor IX (FIX) activity, whereas SaCas9-NNG-mediated editing at all target sites attenuated the FIX activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). These results indicated that SaCas9-NNG exhibits the expanded target scope in mice, and could be used as a therapeutic genome-editing tool deliverable via a single AAV vector.\u003c/p\u003e\n\u003ch3\u003eStructure-guided engineering of the eSaCas9-NNG variant\u003c/h3\u003e\n\u003cp\u003eIn addition to the limited target ranges due to the PAM requirement, off-target effects pose an obstacle to therapeutic applications of CRISPR-based technologies\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To reduce off-target cleavage by SaCas9, we sought to engineer a high-fidelity SaCas9 variant. Previous studies revealed that reducing non-specific interactions between SpCas9 and the DNA backbone improves cleavage fidelity\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In the SaCas9 structure, Asn413 interacts with the ribose moiety of dC19 in the target DNA\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Indeed, the N413A mutation did not alter the on-target activity \u003cem\u003ein vitro\u003c/em\u003e, but reduced the cleavage activity against an off-target DNA containing a single mismatch at the PAM distal end (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). A previous study also revealed that disrupting the salt bridges within the REC domain enhances the fidelity of SpCas9\u003csup\u003e28\u003c/sup\u003e. As observed in SpCas9, the Ala substitution of Arg420, which forms salt bridges with Glu406 and Asp412, reduced off-target cleavage by SaCas9, while maintaining its on-target activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Notably, the N413A/R420A double mutations further enhanced the fidelity of SaCas9, and the inclusion of these mutations also substantially reduced the off-target activity of SaCas9-NNG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The N413A/R420A mutations reduced the cleavage kinetics of both SaCas9 and SaCas9-NNG toward the on-target DNA substrate, as also observed in high-fidelity SpCas9 variants\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo comprehensively assess the effect of the N413A/R420A double mutations on the target specificity, we examined the \u003cem\u003ein vitro\u003c/em\u003e DNA cleavage activities of SaCas9 and SaCas9-NNG toward DNA substrates containing a mismatch at positions 1\u0026ndash;21. While SaCas9 and SaCas9-NNG were tolerant to most single mismatches, the N413A/R420A mutations reduced the activities of SaCas9 and SaCas9-NNG toward mismatch-containing substrates, especially those with PAM-distal mismatches (positions 16, 17, 20, and 21) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Thus, we refer to these high-fidelity variants as enhanced-specificity SaCas9 (eSaCas9) and eSaCas9-NNG, respectively. Furthermore, we compared the on- and off-target activities of eSaCas9 with those of SaCas9-HF (R245A/N413A/N419A/R654A), a rationally engineered high-fidelity SaCas9 variant\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Both variants exhibited similar on- and off-target activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), demonstrating that eSaCas9 possesses comparable cleavage specificity to SaCas9-HF.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eGenome editing by eSaCas9 and eSaCas9-NNG in human cells\u003c/h2\u003e\u003cp\u003eWe compared the genome-editing efficiencies of eSaCas9 and eSaCas9-NNG with those of SaCas9 and SaCas9-NNG at 35 endogenous target sites with NNG PAMs in HEK293T cells. Consistent with our \u003cem\u003ein vitro\u003c/em\u003e data, eSaCas9 and eSaCas9-NNG exhibited comparable genome-editing efficiencies to those of SaCas9 and SaCas9-NNG, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Using GUIDE-seq (genome-wide, unbiased identification of double-stranded breaks enabled by sequencing), we analyzed the genome-wide specificities of SaCas9, eSaCas9, SaCas9-NNG, and eSaCas9-NNG at the \u003cem\u003eVEGFA\u003c/em\u003e site in human cells. As expected, eSaCas9 and eSaCas9-NNG exhibited lower off-target activity than SaCas9 and SaCas9-NNG, respectively (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d). These results demonstrated that the N413A/R420A mutation substantially reduces off-target cleavage by SaCas9, while maintaining on-target activity.\u003c/p\u003e\u003cp\u003eTo evaluate the advantages of eSaCas9-NNG over other Cas9 variants with relaxed PAM constraints, we compared the genome-editing efficiencies of eSaCas9-NNG with those of SpRY\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, SpG\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, and iGeoCas9\u003csup\u003e14,15\u003c/sup\u003e, which recognize NN, NG, and NNNNC as the PAMs, respectively, at six different target sites with NNG PAMs in HEK293T cells (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). eSaCas9-NNG and SpRY induced indels at the six target sites with 14.3% and 8.8% efficiencies on average, respectively, indicating that eSaCas9-NNG outperforms SpRY at target sites with NNG PAMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e). As expected, eSaCas9-NNG, but neither SpG nor iGeoCas9, efficiently induced indels at target sites with NTGNW PAMs (where W is A or T) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e). Moreover, even for targets with NGGNC PAMs, which are equally compatible with the four Cas9 nucleases, eSaCas9-NNG exhibited genome-editing efficiencies comparable to or higher than those of SpG and iGeoCas9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e). Collectively, these findings establish eSaCas9-NNG as a versatile genome-editing tool that can be used for precise gene therapy with a broad target range and robust genome-editing activities.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCryo-EM structure of the eSaCas9-NNG–guide RNA–target DNA complex\u003c/h3\u003e\n\u003cp\u003eTo elucidate the molecular mechanism underlying the relaxed PAM recognition and improved fidelity of eSaCas9-NNG, we determined the cryo-EM structure of eSaCas9-NNG in complex with a 98-nt sgRNA (containing a 21-nt guide) and its target dsDNA containing the TTGCCT PAM at 3.1-\u0026Aring; resolution (Extended Data Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, and Supplementary Table S3,4). eSaCas9-NNG adopts a bilobed architecture consisting of recognition (REC) and nuclease (NUC) lobes, which are connected by a bridge helix (BH) and a linker loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026ndash;c). The NUC lobe consists of the RuvC, HNH, WED, and PI domains, while the REC lobe consists of the REC1 and REC2 domains. Within the NUC lobe, the HNH and RuvC domains are connected by the L1 and L2 linkers. The sgRNA guide segment base-pairs with the TS to form a 21-bp guide RNA\u0026ndash;target DNA heteroduplex, which is accommodated between the REC and NUC lobes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u0026ndash;e). The sgRNA scaffold comprises a repeat:anti-repeat duplex, stem-loop 1, and stem-loop 2 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed,e). The repeat:anti-repeat duplex is sandwiched between the REC1 and WED domains, while stem-loop 1 and stem-loop 2 are recognized by the BH/REC1 and RuvC/PI domains, respectively (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u0026ndash;e). Nucleotides dG(\u0026minus;\u0026thinsp;21*)\u0026ndash;dA(\u0026minus;\u0026thinsp;17*) and dG(\u0026minus;\u0026thinsp;3*)\u0026ndash;dC(\u0026minus;\u0026thinsp;1*) in the single-stranded NTS are bound to the positively charged surfaces of the RuvC and RuvC/L2/PI domains, respectively, while nucleotides dA(\u0026minus;\u0026thinsp;16*)\u0026ndash;dT(\u0026minus;\u0026thinsp;4*) are disordered (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u0026ndash;e). The PAM-containing DNA duplex binds to the surface formed by the WED and PI domains, while nucleotides dA(\u0026minus;\u0026thinsp;25*)\u0026ndash;dC(\u0026minus;\u0026thinsp;22*) in the NTS re-hybridize with nucleotides dG22\u0026ndash;dT25 in the TS to form the PAM-distal DNA duplex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u0026ndash;e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eExcept for the HNH domain, eSaCas9-NNG is structurally similar to the previously reported crystal structure of SaCas9\u003csup\u003e9\u003c/sup\u003e, suggesting that the introduced mutations do not substantially affect the complex structure (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,b). In the SaCas9 crystal structure, the HNH domain is distant from the cleavage site of the TS and interacts with the RuvC domain, indicating that it represents the catalytically inactive state. In contrast, the HNH domain in the present structure docks onto the TS cleavage site and interacts with the REC1 domain and the L1 linker (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,b), and thus represents the catalytically activated state. Indeed, the TS was cleaved between dC3 and dA4 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec,d), although it contains phosphorothioate modifications, possibly due to the high enzyme concentration during reconstitution. The 3\u0026prime;-hydroxy group of dC3 and the phosphate group of dA4 are stabilized by a Mg\u003csup\u003e2+\u003c/sup\u003e ion, which is coordinated by Asp556 and Asn580 in the HNH domain (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec,d). In addition, the dA4 phosphate group is recognized by the catalytic residue His557, corresponding to His840 of SpCas9 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec,d). As in the SaCas9 structure\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, Asp10, Glu477, and His701 in the RuvC domain coordinate two Mg\u003csup\u003e2+\u003c/sup\u003e ions and form the active site responsible for the NTS cleavage (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec,e). These structural observations demonstrated that eSaCas9-NNG cleaves the TS and NTS in Mg\u003csup\u003e2+\u003c/sup\u003e-dependent manners, using the HNH and RuvC nuclease domains, respectively, as observed in other Cas9 orthologs\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eStructural basis for the relaxed PAM recognition by eSaCas9-NNG\u003c/h3\u003e\n\u003cp\u003eThe TTGCCT PAM is recognized by the WED and PI domains in the eSaCas9-NNG structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). As in the SaCas9 structure\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, the third G in the PAM (dG3*) forms bidentate hydrogen bonds with the side chain of Arg1015, which is stabilized by interactions with Glu993 and Phe1017 (I1017F) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). By contrast, the fourth to sixth nucleobases in the PAM (dC4*\u0026ndash;dT6*) lack base-specific interactions with the protein, due to the Ala985 (N985A), Ser986 (N986S), and Ala991 (R991A) replacements (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Notably, the newly incorporated Lys782 (E782K), Lys927 (T927K), and Arg968 (N968R) residues directly interact with the backbone phosphates of the PAM duplex, while Arg800 (L800R) and Ser1021 (A1021S) likely form water-mediated hydrogen bonds with the PAM duplex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In addition, Asn929 (K929N) stabilizes the conformation of Lys927, which interacts with the backbone phosphate of dG3* (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Accordingly, these newly formed non-base-specific interactions compensate for the loss of base-specific interactions with the fourth to sixth PAM nucleobases, thereby achieving the relaxed NNG PAM recognition by SaCas9-NNG. Whereas Arg1015 hydrogen bonds with the fourth G in the NNGG PAMs in the SaCas9 structure\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg), Arg1015 is slightly farther away from the fourth PAM nucleobase in the eSaCas9-NNG structure, due to the presence of the introduced Phe1017, preventing the interaction between Arg1015 and the fourth G (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). These structural observations account for the reduced activity of eSaCas9-NNG toward DNA substrates with NNGG PAMs.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStructural basis for the improved specificity of eSaCas9-NNG\u003c/h2\u003e\u003cp\u003eTo understand the structural basis for the improved specificity of eSaCas9-NNG, we determined the cryo-EM structures of SaCas9 and eSaCas9-NNG in complex with an sgRNA and its target dsDNA containing a single-nucleotide mismatch at position 21 from the PAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c, and Extended Data Fig.\u0026nbsp;7). SaCas9 and eSaCas9-NNG adopt almost identical overall structures, indicating that the N413A/R420A mutations do not alter the overall conformation of the protein when bound to a mismatched target (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Notably, both structures represent the catalytically active conformation where the HNH domain docks onto the TS cleavage site, suggesting that the N413A/R420A mutations in the REC2 domain do not directly regulate the HNH domain conformation in the presence of a PAM-distal mismatch, in contrast to the allosteric regulation between the REC3 and HNH domains observed in high-fidelity variants of SpCas9\u003csup\u003e27\u003c/sup\u003e. The PAM-distal G1:dG21 mismatch likely forms a Hoogsteen base pair in both SaCas9 and eSaCas9-NNG, resulting in the slight distortion of the TS backbone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee,f and Extended Data Fig.\u0026nbsp;8a,b).\u003c/p\u003e\u003cp\u003eWhile the overall structures of SaCas9 and eSaCas9-NNG are similar, we observed a conformational difference in a helix of the REC2 domain (residues 414\u0026ndash;421, referred to as the mismatch-sensing helix (MSH)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u0026ndash;g and Extended Data Fig.\u0026nbsp;8c,d). In the SaCas9 structure, the MSH interacts with the PAM-distal TS backbone, with Asn413 and Asn419 forming hydrogen-bonding interactions with dC19 and dC20, respectively. Notably, Arg420 forms salt bridges with Glu406 and Asp412, stabilizing the relative position of the MSH. These interactions facilitate the stable binding of the mismatch-containing distorted TS to SaCas9, thereby enabling the efficient cleavage of a target DNA with a PAM-distal mismatch. In contrast, in the eSaCas9-NNG structure, the Asn413\u0026ndash;dC19 hydrogen bond and the Glu406\u0026ndash;Arg420\u0026ndash;Asp412 salt bridge network are eliminated due to the N413A and R420A mutations, leading to an outward displacement of the MSH from the TS backbone. Furthermore, in the eSaCas9-NNG structure, the side chain of Asn419 adopts a flipped-out conformation and disrupts the Asn419\u0026ndash;dC20 hydrogen bond. These local structural changes cause further distortion of the PAM-distal TS backbone, likely destabilizing the active-state conformation of the protein and impairing the ability of eSaCas9-NNG to efficiently cleave DNA with a PAM-distal mismatch (Supplementary Movie S1). Collectively, these structural observations suggest that the target specificity of SaCas9 is regulated by the interactions between the MSH and the PAM-distal TS backbone.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCryo-EM structures of eSaCas9-NNG in distinct functional states\u003c/h2\u003e\u003cp\u003eIn addition to the catalytically active state (State I), our cryo-EM analysis revealed three distinct classes (States II\u0026ndash;IV), which are primarily distinguished by the guide RNA\u0026ndash;target DNA heteroduplex lengths (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We therefore determined the three additional structures at overall resolutions of 3.2 \u0026Aring; (State II), 2.9 \u0026Aring; (State III), and 2.8 \u0026Aring; (State IV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026ndash;c and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn State II, the sgRNA guide does not hybridize with the double-stranded target DNA, which instead binds to the groove between the WED and PI domains, with the third G PAM nucleotide recognized by Arg1015 in the PI domain, as in the catalytically active state (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,d). Thus, this structure likely represents the \u0026ldquo;interrogation state\u0026rdquo;, in which the Cas9\u0026ndash;sgRNA complex recognizes a PAM sequence but has not yet unwound the double-stranded target DNA to hybridize with the TS, as previously observed in the cryo-EM structure of SpCas9 in the interrogation state, which was artificially stabilized by a protein\u0026ndash;DNA cross-link\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In SpCas9 and SaCas9, the WED, RuvC, HNH, and PI domains are structurally ordered in this state and adopt similar conformations to those in the inactive state\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In contrast, the BH, REC1, and REC2 domains are disordered in the interrogation state of SaCas9, suggesting that they are flexible before the heteroduplex formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Notably, nucleotides G15\u0026ndash;C21 in the sgRNA are pre-ordered in an A-form geometry for base-pairing with the TS, even without interactions with the BH (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eIn State III, nucleotides A4\u0026ndash;C21 in the sgRNA base-pair with nucleotides dG1\u0026ndash;dT18 in the TS to form an 18-bp heteroduplex, while the three PAM-distal nucleotides (G1\u0026ndash;G3) in the sgRNA are disordered (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). This structure represents the \u0026ldquo;intermediate state\u0026rdquo;, in which the Cas9\u0026ndash;sgRNA complex has partially formed the guide\u0026ndash;target heteroduplex. In this state, the BH and the REC1 domain become ordered and extensively interact with the PAM-proximal region of the heteroduplex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). In contrast, the REC2 domain remains disordered, with the PAM-distal region of the heteroduplex exposed to the solvent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The HNH domain is located far from the TS cleavage site, while the RuvC active site is occluded by the L1 linker, as in the interrogation state (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b), indicating that the formation of the 18-bp heteroduplex is insufficient to activate SaCas9, as previously reported\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn State IV, nucleotides G1\u0026ndash;C21 in the sgRNA base-pair with nucleotides dG1\u0026ndash;dC21 in the TS to form the complete 21-bp heteroduplex, as in the catalytically active state (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In this state, the REC2 domain becomes ordered and interacts with the PAM-distal region of the heteroduplex, while the HNH domain, along with the L1 and L2 linkers, becomes disordered (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Thus, this structure represents a \u0026ldquo;translocation state\u0026rdquo;, in which the HNH domain is moving toward the TS cleavage site. Although the dissociation of the L1 linker allows the NTS to access the RuvC domain, unlike the catalytically active state, only one Mg\u003csup\u003e2+\u003c/sup\u003e ion is bound to the RuvC active site (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef), indicating that the RuvC domain does not adopt a cleavage-competent active conformation in the translocation state.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eNuclease activation mechanism\u003c/h2\u003e\u003cp\u003eThese four structures in different functional states provide mechanistic insights into the dynamic nuclease activation of SaCas9. A structural comparison of the interrogation and intermediate states reveals that the ordering of the BH and the REC1 domain is coupled with the shift of the pre-ordered guide region (G15\u0026ndash;C21) toward the interior of the protein, resulting in the formation of the 18-bp guide\u0026ndash;target heteroduplex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). A structural comparison between the intermediate and translocation states demonstrates that the ordering of the REC2 domain is coupled with the formation of the 21-bp guide\u0026ndash;target heteroduplex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb,c). The heteroduplex elongation facilitates a conformational change in the L2 linker (residues 629\u0026ndash;649) (Extended Data Fig.\u0026nbsp;9a\u0026ndash;f). In the intermediate state, residues 635\u0026ndash;645 in the L2 linker form an α helix and interact with both the RuvC and HNH domains, stabilizing the inactive conformation (Extended Data Fig.\u0026nbsp;9a,d). In contrast, in the translocation state, this α helix is structurally melted and the HNH domain becomes disordered (Extended Data Fig.\u0026nbsp;9b,e). A structural comparison of the two states suggests a steric clash between the L2 α helix in the intermediate state and the heteroduplex in the translocation state (Extended Data Fig.\u0026nbsp;9c,f), indicating that the formation of the 21-bp heteroduplex induces the structural rearrangement of the L2 linker, resulting in the dissociation of the HNH domain from the RuvC domain (Extended Data Fig.\u0026nbsp;9a\u0026ndash;f). In the catalytically active state, the HNH domain undergoes an approximately 160\u0026deg; rotation from its inactive position and docks onto the TS cleavage site in the heteroduplex (Extended Data Fig.\u0026nbsp;9g,h). This HNH rearrangement is accompanied by structural changes in the REC1 domain and the L1/L2 linkers. Whereas residues 126\u0026ndash;146 in the REC1 domain are located near the TS cleavage site in the intermediate and translocation states, these residues become disordered in the active state, due to the docking of the HNH domain (Extended Data Fig.\u0026nbsp;9h). The L1 linker undergoes an approximately 180\u0026deg; rotation from its position in the intermediate state and binds to the minor groove of the PAM-distal heteroduplex (Extended Data Fig.\u0026nbsp;9i,j). This L1 rearrangement is accompanied by a structural change in the β4 strand (Glu477) of the RuvC domain, thus forming the RuvC active site (Extended Data Fig.\u0026nbsp;9k). In the active state, the L2 linker adopts a loop conformation to form an NTS-binding pathway toward the RuvC active site (Extended Data Fig.\u0026nbsp;9j). In particular, Phe635 in the L2 linker stacks with the dA(\u0026minus;\u0026thinsp;1)-dT1* base pair in the PAM duplex, while Arg1002 in the PI domain interacts with the flipped-out dC(\u0026minus;\u0026thinsp;1*) in the NTS (Extended Data Fig.\u0026nbsp;9l). Collectively, these structural observations revealed the coordinated domain rearrangements coupled with the formation of the guide\u0026ndash;target heteroduplex to achieve target DNA cleavage by SaCas9 (Supplementary movies S2 and S3).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSaCas9 was identified in 2015 as the first compact Cas9, and has since been used as a versatile genome editing tool in human cells and various organisms\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. However, the development of useful SaCas9 variants has been limited as compared to SpCas9, and the action mechanism of the small SaCas9 has remained enigmatic. In this study, we rationally engineered the eSaCas9-NNG variant with an expanded targeting range and reduced off-target activity. We also determined the cryo-EM structures of eSaCas9-NNG in four distinct states, illuminating the dynamic activation mechanism of the small SaCas9 enzyme.\u003c/p\u003e\u003cp\u003eRecent studies revealed coordinated conformational rearrangements of multiple domains, including the REC2/REC3 and HNH domains, coupled with the guide–target heteroduplex formation in target DNA cleavage by SpCas9\u003csup\u003e31,33,34,38\u003c/sup\u003e. However, Cas9 orthologs have structurally diverse REC lobes, and small Cas9 enzymes, such as SaCas9, lack a domain equivalent to the REC2 domain of SpCas9 (the REC2 domain of SaCas9 corresponds to the REC3 domain of SpCas9). Therefore, the activation mechanisms of small Cas9 orthologs have remained elusive. The eSaCas9-NNG structures in the four different states illuminated the conformational rearrangements coupled with the guide–target heteroduplex formation, highlighting the mechanistic conservation of Cas9 nuclease activation. In the inactive states of both SpCas9 and SaCas9, the HNH domain is located far from the TS cleavage site (Extended Data Fig.\u0026nbsp;10a,b). Upon the guide–target heteroduplex formation and the accompanying REC rearrangement, the HNH domains of SpCas9 and SaCas9 undergo rotations of approximately 140° and 160°, respectively, docking onto the TS cleavage site (Extended Data Fig.\u0026nbsp;10c,d). In both SpCas9 and SaCas9, the L1 and L2 linkers participate in recognizing the PAM-distal region of the heteroduplex and guiding the NTS toward the RuvC active site, respectively (Extended Data Fig.\u0026nbsp;10e,f). These structural observations suggest that the inactive-to-active HNH domain rearrangements via the L1/L2 linkers are conserved among Cas9 activation mechanisms.\u003c/p\u003e\u003cp\u003eWe also found notable differences in the activation mechanisms between SpCas9 and SaCas9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In SpCas9, the entire REC lobe is ordered when the 6-bp guide–target heteroduplex forms, creating a positively charged cleft that accommodates the PAM-distal DNA duplex\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. By contrast, in SaCas9, while the REC1 domain is ordered, the REC2 domain remains disordered even when the 18-bp guide–target heteroduplex forms. This difference is likely attributed to the absence of REC1–REC2 interactions and the presence of the larger WED domain in SaCas9, as compared to SpCas9. Furthermore, whereas the formation of a 12-bp heteroduplex induces a structural change in the HNH domain in SpCas9\u003csup\u003e33\u003c/sup\u003e, the formation of an 18-bp heteroduplex in SaCas9 does not induce the HNH rearrangement. This variation is likely due to the structural differences in the HNH domains and the L1/L2 linkers between SpCas9 and SaCas9. Collectively, the structurally divergent REC lobes and L1/L2 linkers contribute to the distinct activation mechanisms of the Cas9 orthologs, which may result in their different cleavage efficiencies and fidelities.\u003c/p\u003e\u003cp\u003eMoreover, our cryo-EM structures revealed that the structurally divergent REC lobes also contribute to the distinct mismatch-sensing mechanisms of the Cas9 orthologs. In SpCas9, the REC3 domain senses the integrity of the PAM-distal region of the guide–target heteroduplex and allosterically regulates the HNH domain movement by reorienting the REC2 domain\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. These allosteric domain rearrangements establish a conformational checkpoint that traps SpCas9 in an inactive state when bound to a mismatched target. Indeed, single molecule experiments have demonstrated that HNH domain docking onto the TS is completely abolished when some high-fidelity SpCas9 variants bind to mismatched targets\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In contrast, SaCas9 lacks REC1–REC2 interactions and employs a distinct mismatch-sensing mechanism, wherein the MSH in the REC2 domain detects PAM-distal mismatches and presumably affects the stability of the active conformation of the HNH domain, thereby governing its target specificity.\u003c/p\u003e\u003cp\u003eWhile SpCas9 efficiently cleaves its target DNA using a 20-nt guide sgRNA, most small Cas9 orthologs require 1–2-nt longer guides for efficient DNA cleavage (\u003cem\u003ee.g.\u003c/em\u003e, 21- and 22-nt guides are optimal for SaCas9 and CjCas9, respectively)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Although the choice of optimal guide lengths is an important factor in Cas9-mediated genome engineering, it remains unclear why the Cas9 orthologs require different guide lengths for efficient DNA cleavage. A structural comparison between SpCas9 and SaCas9 provides an explanation for their different optimal guide lengths. In the active state of SpCas9, Arg765 in the L1 linker interacts with the backbone phosphate of the 5′ end (G1) of the 20-nt guide\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e (Extended Data Fig.\u0026nbsp;10g). In the SaCas9 structure, Arg480 and Lys482 in the L1 linker interact with the backbone phosphate of G1 in the 21-nt guide (Extended Data Fig.\u0026nbsp;10h). These interactions between Cas9s and the 5′ ends of their guides are consistent with their optimal guide lengths. Although further elongation of the guide–target heteroduplex seems possible without steric clashes, the PAM-proximal end of the heteroduplex is fixed within the central groove of SaCas9, likely resulting in an energetically unfavorable supercoiling effect with extended base pairing outside the SaCas9 complex.\u003c/p\u003e\u003cp\u003eIn summary, we developed the eSaCas9-NNG variant, which will expand the CRISPR toolbox for \u003cem\u003ein vivo\u003c/em\u003e therapeutic genome editing. In addition, our cryo-EM analysis provides structural snapshots of the small SaCas9 during target DNA cleavage, improving our mechanistic understanding of diverse CRISPR-Cas9 enzymes. To date, structure-guided Cas9 engineering has primarily relied on crystal structures captured in the inactive state. By leveraging active- and intermediate-state structures, such as those resolved in this study, future Cas9 engineering efforts can be guided by a more rational and precise structural framework, enabling the development of next-generation genome editors with enhanced specificity and efficiency.\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Methods","content":"\u003ch2\u003eProtein and RNA preparation for structural analysis\u003c/h2\u003e\u003cp\u003eThe gene encoding full-length SaCas9 (residues 1–1,053) was codon optimized, synthesized (Genscript), and cloned between the \u003cem\u003eNde\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI sites of the modified pE-SUMO vector (LifeSensors). The mutations were introduced by a PCR-based method, using the vector encoding full-length SaCas9 as the template, and the sequences were confirmed by DNA sequencing (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The SaCas9 protein was expressed and purified using the protocol reported previously\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Briefly, the N-terminally His\u003csub\u003e6\u003c/sub\u003e-tagged SaCas9 proteins were expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e Rosetta2 (DE3). The SaCas9-expressing \u003cem\u003eE. coli\u003c/em\u003e cells were cultured at 37°C in LB medium (containing 20 mg/L kanamycin) until the OD\u003csub\u003e600\u003c/sub\u003e reached 0.8, and protein expression was then induced by the addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside (Nacalai Tesque). The \u003cem\u003eE. coli\u003c/em\u003e cells were further cultured at 20°C for 18 hr, and harvested by centrifugation at 5,000 g for 10 min. The \u003cem\u003eE. coli\u003c/em\u003e cells were resuspended in buffer A (20 mM Tris-HCl, pH 8.0, 20 mM imidazole, and 1 M NaCl), lysed by sonication, and then centrifuged at 10,000 g for 20 min. The supernatant was mixed with 0.3 mL Ni-NTA Superflow resin (QIAGEN) equilibrated with buffer A, and the mixture was loaded into a Poly-Prep Column (Bio-Rad). The protein was eluted with buffer B (20 mM Tris-HCl, pH 8.0, 300 mM imidazole, and 300 mM NaCl). To remove the His\u003csub\u003e6\u003c/sub\u003e-SUMO-tag, the eluted protein was mixed with SUMO protease, and then dialyzed at 4°C overnight against buffer C (20 mM Tris-HCl, pH 8.0, and 300 mM NaCl). The protein was loaded onto a HiTrap SP HP column (GE Healthcare) equilibrated with buffer C, and eluted with a linear gradient of 0.3–2 M NaCl. The protein was further purified by chromatography on a HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated with buffer D (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 1 mM DTT). The purified proteins were stored at − 80°C until use. The sgRNA was transcribed \u003cem\u003ein vitro\u003c/em\u003e with T7 RNA polymerase, using a partially double-stranded DNA template. The transcribed sgRNA was purified by 8% denaturing urea PAGE, extracted from gel slices with Tris-Borate-EDTA Buffer (Takara), and then ethanol precipitated. The sgRNA pellet was dissolved in nuclease-free water and stored at − 20°C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecleavage assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe linearized pUC119 plasmid (100 ng, 4.7 nM), containing the 23-nt target sequence and the PAMs (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), was incubated at 37°C for 0.5–5 min with the SaCas9–sgRNA complex (50 nM) in 10 µL of reaction buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT, and 5% glycerol). The reactions were stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng). The reaction products were resolved, visualized, and quantified with a MultiNA microchip electrophoresis device (SHIMADZU).\u003c/p\u003e\u003ch2\u003ePAM identification assay\u003c/h2\u003e\u003cp\u003eThe PAM identification assay was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The PAM library (100 ng), containing eight randomized nucleotides downstream of a 21-nt target sequence, was incubated at 37°C for 5 min with the purified SaCas9 (SaCas9 and SaCas9-NNG) (50 nM) and the sgRNA (21-nt guide) in 10 µL of reaction buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT, and 5% glycerol). The reactions were stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng), and then purified using a Wizard DNA Clean-Up System (Promega). The purified DNA samples were amplified for 25 cycles, using primers containing common adapter sequences. After column purification, each PCR product (~ 5 ng) was subjected to a second round of PCR for 15 cycles, to add custom Illumina TruSeq adapters and sample indices. The sequencing libraries were quantified by qPCR (KAPA Biosystems), and then subjected to paired-end sequencing on a MiSeq sequencer (Illumina) with 20% PhiX spike-in (Illumina). The sequencing reads were demultiplexed by primer sequences and sample indices, using NCBI Blast + (version 2.8.1) with the blastn-short option. For each sequencing sample, the number of reads for every possible 8-nt PAM sequence pattern (4\u003csup\u003e8\u003c/sup\u003e = 65,536 patterns in total) was counted and normalized by the total number of reads in each sample. For a given PAM sequence, the enrichment score was calculated as log\u003csub\u003e2\u003c/sub\u003e-fold enrichment as compared to the untreated sample. PAM sequences with enrichment scores of − 2.0 or less were used to generate the sequence logo representation, using WebLogo (version 3.7.1)\u003csup\u003e43\u003c/sup\u003e. The cumulative distribution and histogram of the read count of each PAM in the unedited sample confirmed that the plasmid library has sufficient coverage for the individual PAM sequences.\u003c/p\u003e\u003ch2\u003eGenome- and base-editing analyses in human cells\u003c/h2\u003e\u003cp\u003eGenome- and base-editing analyses were performed, according to the protocol described previously\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Briefly, HEK293Ta cells were maintained in DMEM (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Sigma), at 37°C in a 0.05% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. HEK239Ta cells were seeded at 5×10\u003csup\u003e3\u003c/sup\u003e cells per well in collagen I-coated 96-well plates, 24 hr prior to transfection. HEK239Ta cells were transfected with a SaCas9 plasmid or a SaCas9-derived base-editor plasmid (120 ng) and an sgRNA plasmid (40 ng), using Polyethylenimine Max (Polysciences) (1 mg/mL, 0.5 µL) in PBS (50 µL) (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The cells were harvested 3 days after transfection, treated with 50 mM NaOH (100 µL), incubated at 95°C for 10 min, and then neutralized with 1 M Tris-HCl, pH 8.0 (10 µL). The obtained genomic DNA was subjected to two rounds of PCR, to prepare the library for high-throughput amplicon sequencing. Genomic regions targeted by sgRNAs were PCR-amplified to add custom primer-landing sequences. The PCR products were purified by AMPure XP magnetic beads (Agencourt), and then subjected to a second round of PCR to attach the custom Illumina TruSeq adapters with sample indices. After size-selection by agarose gel electrophoresis and column purification, the sequencing libraries were quantified using a KAPA Library Quantification Kit Illumina (KAPA Biosystems), multiplexed, and subjected to paired-end sequencing (600 cycles), using a MiSeq sequencer (Illumina) with 20% PhiX spike-in (Illumina). The sequencing reads were demultiplexed, based on sample indices and primer sequences. Using NCBI BLAST + (version 2.6.0) with the blastn-short option, the sequencing reads were mapped to the reference sequences to identify indels and substitutions in the target regions. To remove common PCR errors and somatic mutations, we deleted sequencing reads containing mutations (\u0026gt; 1% frequency) commonly observed in the control samples from the edited samples, and then normalized the editing frequencies for the target sites by subtracting the mutation frequencies of the control samples from those of the edited samples.\u003c/p\u003e\u003ch2\u003eComparison of genome-editing efficiencies among various Cas9 variants in human cells\u003c/h2\u003e\u003cp\u003eHEK293T cells were maintained in DMEM (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS) (Nichirei), 1% GlutaMAX (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Sigma), at 37°C in a 0.05% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Cells (5×10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in 48 well plates coated with collagen type I (Cellmatrix type I-C, Nitta Gelatin) the day before transduction. The plasmids (200 ng) were incubated together with Lipofectamine 3000 (Thermo Fisher Scientific), and then directly added to the cell culture according to the manufacturer's recommendations. At 48 h after the transduction, the cells were lysed with the SimplePrep reagent for DNA (Takara Bio). The supernatants were directly used for PCR. DNA fragments were amplified with Phusion DNA polymerase (New England Biolabs). PCR amplicons were subjected to 150-bp pair-end read sequencing using the Illumina MiSeq at Genome-Lead Co., Ltd. (Kagawa, Japan). The frequencies of the mutations were assessed by CRISPResso2\u003csup\u003e45\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eGUIDE-Seq analysis\u003c/h2\u003e\u003cp\u003eHEK293 and U2OS cells were maintained and cultured as described previously\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Cells were nucleofected following the manufacturer’s instructions (Lonza) in 20 µL Solution SE, using programs CM-104 (293) and DN-100 (U2OS) on a Lonza Nucleofector 4-D. Cells were transfected with SaCas9 plasmids (500 ng), sgRNA plasmids (250 ng), and dsODN [100 pmol; complementary oligonucleotide sequences derived from Malinin et al. 2021\u003csup\u003e47\u003c/sup\u003e].\u003c/p\u003e\u003cp\u003eGUIDE-seq library preparation and analysis were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Briefly, genomic DNA was purified via an Agencourt DNAdvance kit (Beckman Coulter). A Covaris E220 ultrasonicator was used to shear purified genomic DNA to an average fragment size of 500 bp. After sonication, 400 ng was used for library preparation. Genomic DNA was treated with end-repair mix (Qiagen), A-tailed with Taq polymerase (Thermo Fisher Scientific), ligated to single-tailed sequencing adapters, and purified using SPRI magnetic beads. Two rounds of nested PCR with the dsODN sense- and antisense-specific primers in separate reactions were performed on the adapter-ligated library. After purification with SPRI magnetic beads, libraries were quantified using the Kapa qPCR Library Quantification Kit (Kapa). Equimolar amounts of samples were pooled and sequenced with 150 bp paired end reads on the Illumina NextSeq 550 sequencer.\u003c/p\u003e\u003cp\u003eFor GUIDE-seq-2 library preparation, Tn5 transposase was prepared by combining hyperactive Tn5 with annealed i5 adapter oligos containing an 8-nucleotide barcode and 10-nucleotide unique molecular index, in 2x Tn5 dialysis buffer (100 mM HEPES-KOH pH 7.2, 200 mM NaCl, 0.2 mM EDTA, 2 mM DTT, 0.2% Triton X-100, and 20% glycerol), for one hr at 24°C. Tagmentation was performed in 40 µL reactions for 7 min at 55°C, using 250 ng of genomic DNA, 4 µL of assembled Tn5/i5-transposome, and 8 µL of fresh 5x TAPS-DMF buffer (50 mM TAPS-NaOH, 25 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 50% dimethylformamide (DMF)). To stop the reaction, 5 µL of a 50% proteinase K (NEB) solution was added, and the solution was incubated for 15 min at 55°C. Samples were purified using SPRI-guanidine magnetic beads, and separate PCR reactions were performed using dsODN sense- and antisense-specific primers. Reactions were conducted with Platinum Taq (Thermo Fisher) using the following thermocycler settings: 95°C for 5 min, 15 cycles of temperature cycling (95°C for 30 sec, 70°C (-1°C per cycle) for 120 sec, and 72°C for 30 sec), 20 constant cycles (95°C for 30 sec, 55°C for 60 sec, and 72°C for 30 sec), and 72°C for 5 min. PCR products were purified using SPRI beads and quantified using a Kapa qPCR Library Quantification Kit (Kapa). Libraries were purified using Lightbench (Yourgene Health) selection, and sequenced using a NextSeq1000/2000 (Illumina) sequencer with cycle settings of 146, 8, 18, and 146. Data analysis was performed using the updated open-source GUIDE-seq2 analysis software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tsailabSJ/guideseq/tree/V2\u003c/span\u003e\u003cspan address=\"https://github.com/tsailabSJ/guideseq/tree/V2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003ch2\u003ePlasmid construction, AAV production, and assessment of genome editing\u003c/h2\u003e\u003cp\u003eAAVpro293T cells (Takara Bio) and HEK293 cells were cultured in DMEM (Sigma) supplemented with 10% FBS (Thermo Fisher Scientific) and GlutaMAX (Thermo Fisher Scientific). Murine immortalized liver cells (TLR3 cells, JCRB Cell Bank) were maintained in DMEM containing 2% FBS, 5 ng/mL of human epidermal growth factor (EGF), and ITS-X Supplement (Thermo Fisher Scientific). The SaCas9 cDNA was codon-optimized in GenScript. A DNA fragment comprising a promoter, SaCas9 cDNA (SaCas9 or SaCas9-NNG), the SV40 polyadenylation signal, and sgRNA sequence driven by the U6 promoter was introduced into the p1.1c plasmid. The HCRhAAT liver-tropic promoter (an enhancer element of the hepatic control region of the ApoE/C1 gene and the human anti-trypsin promoter) was employed. SaCas9-ABE8e and the human coagulation factor IX (FIX) cDNA were introduced into the pcDNA3 (Thermo Fisher Scientific) and pBApo-EF1α Neo (Takara) vectors, respectively. The sgRNA driven by the U6 promoter was incorporated into pUC57. The DNA fragment for the SaCas9 expression cassette was introduced between the inverted terminal repeats of the pAAV plasmid. The AAV genes were packaged by triple plasmid transfection of AAVpro293T cells to produce the AAV vector (helper-free system), as described previously\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The titers of recombinant AAV vectors were determined by quantitative PCR, as previously described\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCells (5×10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in 48 well plates coated with collagen type I (Cellmatrix type I-C, Nitta Gelatin) the day before transduction. The plasmids (200 ng) were incubated together with Lipofectamine 3000 (Thermo Fisher Scientific), and then directly added to the cell culture according to the manufacturer's recommendations. To obtain stable expressing clones, 400 µg/mL G418 (Nacalai Tesque) was added to the culture medium after the transfection with pBApo-EF1α Neo. The DNAs were isolated at 72 hr after the transduction.\u003c/p\u003e\u003cp\u003eDNA fragments at the target site were amplified with ExTaq DNA polymerase (Takara Bio). Purified PCR products were denatured and re-annealed using a thermal cycler, and then treated with T7 endonuclease (Nippon Gene). DNA fragments were analyzed by a MultiNA microchip electrophoresis system. When indicated, PCR amplicons were subjected to 300-bp paired-end read sequencing at the NGS core facility at the Research Institute for Microbial Diseases of Osaka University (Osaka, Japan). The mutation frequencies were assessed by CRISPResso2\u003csup\u003e45\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eAnimal experiments\u003c/h2\u003e\u003cp\u003eAll animal experimental procedures were approved by The Institutional Animal Care and Concern Committee of Jichi Medical University (permission number: 20051-10), and animal care was conducted in accordance with the committee’s guidelines and ARRIVE guidelines\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. C57BL/6 mice were purchased from SLC Japan (Shizuoka, Japan). The AAV vector was administered intravenously through the jugular vein (100–150 µL) of mice anesthetized with isoflurane (1–3%). To obtain plasma samples, blood samples were drawn from the jugular vein using a 29G micro-syringe (TERUMO) containing 1/10 (volume/volume) sodium citrate. Platelet-poor plasma was obtained by centrifugation and then frozen and stored at − 80°C until analysis. Plasma FIX activity (FIX:C) was measured by a one-stage clotting-time assay with an automated coagulation analyzer (Sysmex CS-1600).\u003c/p\u003e\u003ch2\u003eCryo-EM sample preparation and data collection\u003c/h2\u003e\u003cp\u003eThe eSaCas9-NNG–sgRNA–target DNA ternary complex was reconstituted by mixing the purified eSaCas9-NNG, the 98-nt sgRNA, the 43-nt target DNA, and the 43-nt non-target DNA at a molar ratio of 1:1.2:1.25:1.25 at room temperature for 10 min. Each DNA strand contained phosphorothioate modifications within the phosphate backbone around the cleavage site to prevent DNA cleavage (Supplementary Table S3). The SaCas9 and eSaCas9-NNG in complex with the sgRNA and target dsDNA containing the single-nucleotide mismatch at position 21 were reconstituted in the same way, except that the incubation was performed at room temperature for 10 min. The ternary complexes were purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare), equilibrated with buffer E (20 mM HEPES-NaOH, pH 7.6, 50 mM NaCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 µM ZnCl\u003csub\u003e2\u003c/sub\u003e, and 1 mM DTT). The purified complex solution (A\u003csub\u003e260 nm\u003c/sub\u003e = 26) was mixed with 0.005% Tween 20, and then applied to Au 300-mesh R1.2/1.3 grids (Quantifoil) that were glow-discharged in a Vitrobot Mark IV (FEI) at 4°C, with a waiting time of 10 sec and a blotting time of 4 sec under 100% humidity conditions. The grids were plunge-frozen in liquid ethane and cooled to the temperature of liquid nitrogen.\u003c/p\u003e\u003cp\u003eMicrographs for all datasets were collected with a Titan Krios G3i microscope (Thermo Fisher Scientific) running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode (The University of Tokyo, Japan). Datasets were collected with a total dose of approximately 50 electrons per Å\u003csup\u003e2\u003c/sup\u003e per 48 frames by the standard mode, using the EPU software (Thermo Fisher Scientific). The dose-fractionated movies were subjected to beam-induced motion correction and dose weighting using Patch Motion Correction, and the contrast transfer function (CTF) parameters were estimated using Patch-based CTF estimation in cryoSPARC v4.4.0\u003csup\u003e52,53\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eCryo-EM data processing\u003c/h2\u003e\u003cp\u003eData were processed with the cryoSPARC v4.4.0 software platform\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. For the eSaCas9-NNG–sgRNA–target DNA ternary complex, 4,900,536 particles were automatically picked using Template Picker from the 8,625 motion-corrected and dose-weighted micrographs, followed by several rounds of reference-free 2D classification to curate particle sets. Using maps derived from ab-initio reconstruction as templates, 1,659,239 selected particles were subjected to heterogeneous refinement, resulting in the reconstructions of six distinct conformational states. Four of these classes, corresponding to State I (catalytically active state), State II (interrogation state), State III (intermediate state), and State IV (translocation state), were subjected to further processing. For States I, III, and IV, the particles were subjected to CTF refinement, Reference-Based Motion Correction (RBMC), and 3D classification without alignment. Non-uniform refinement after subsequent postprocessing yielded maps at overall resolutions of 3.14 Å (catalytically active state), 2.90 Å (intermediate state), and 2.76 Å (translocation state), according to the Fourier shell correlation (FSC) criterion of 0.143\u003csup\u003e54,55\u003c/sup\u003e. For the interrogation state, the selected particles were subjected to 3D variability analysis\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The resulting maps with different conformations were used for subsequent heterogeneous refinement. The particles with the most detailed features after heterogeneous refinement were refined using non-uniform refinement after CTF refinement and RBMC, and yielded a map at an overall resolution of 3.17 Å according to the FSC criterion of 0.143. The local resolution was estimated by BlocRes in cryoSPARC.\u003c/p\u003e\u003cp\u003eThe datasets for SaCas9 and eSaCas9-NNG in complex with the sgRNA and the mismatched target dsDNA were processed using cryoSPARC in a similar manner as described above. For data processing details, see Extended Data Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eModel building and validation\u003c/h2\u003e\u003cp\u003eThe models were built using the crystal structure of SaCas9 (PDB: 5CZZ) as the reference\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, followed by manual model building with Coot\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The models were refined using Servalcat against unsharpened half maps\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The reference structure restraints were used for the refinement of the catalytically active, interrogation, and intermediate states, which were generated from the AlphaFold2 predicted models and the intermediate and translocation state models using ProSmart\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. The stereochemical restraints for phosphorothioate modified DNA links were generated using AceDRG\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The models were validated using MolProbity\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Molecular graphics figures were prepared with UCSF ChimeraX-1.7.1\u003csup\u003e64\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eQUANTIFICATION AND STATISTICAL ANALYSIS\u003c/h2\u003e\u003cp\u003eNo statistical methods were used to predetermine sample size. Sample size was based on experimental feasibility and sample availability. Samples were processed in random order. Statistical analyses were performed using GraphPad Prism 10 (Graph Pad Software, San Diego, CA). All data are presented as the mean ± standard deviation (s.d.).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Sachiyo Kamimura and Yuiko Ogihara of Jichi Medical University and Keiko Ogomori of the University of Tokyo for their technical assistance. T.O. was supported by AMED Grant Numbers JP23fk0410037, JP24fk0410061, JP24bm1323001, and JP23ae0201007. H.N. is supported by JSPS KAKENHI Grant Numbers 21H05281 and 22H00403, the Takeda Medical Research Foundation, the Inamori Research Institute for Science, and JST, CREST Grant Number JPMJCR23B6. O.N. was supported by AMED Grant Numbers JP23fa627001 and JP19am0401005, the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED, under grant numbers JP23ama121002 (support number 3272, M.K.) and JP23ama121012 (support no. 4894, O.N.), and the Cabinet Office, Government of Japan, Public/Private R\u0026amp;D Investment Strategic Expansion Program (PRISM) Grant Number JPJ008000.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.N.O., R.N., S.K., and H.N. performed biochemical experiments with assistance from S.O. and K.H.; S.I., H.M., S.N.O. and N.Y. performed cell biological experiments with assistance from M.T. and K.H.; Y.K., T.H., and T.O. conducted AAV preparation and mouse experiments; K.J. and S.Q.T. conducted GUIDE-seq analysis; S.N.O. and R.N. performed structural analyses with assistance from H.H., K.Y., Y.I., and H.N.; S.N.O., R.N., H.N., and O.N. wrote the manuscript with help from all authors; H.N. and O.N. supervised the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA patent application has been filed related to this work.\u0026nbsp;O.N. is a co-founder, board member, and scientific advisor of Curreio. The remaining authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLead Contact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Osamu Nureki ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and code availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe atomic models of eSaCas9-NNG–guide RNA–target DNA complexes have been deposited in the Protein Data Bank under the accession codes 8ZCY (interrogation state), 8ZCZ (intermediate state), 8ZD0 (translocation state), and 8ZDA (active state). The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-39941 (interrogation state), EMD-39942 (intermediate state), EMD-39944 (translocation state), and EMD-39954 (active state). The atomic models of SaCas9 and eSaCas9-NNG bound to the sgRNAs and target DNA containing a mismatch at position 21 have been deposited in the Protein Data Bank under the accession codes 9MB6 (SaCas9) and 9MB7 (eSaCas9-NNG). The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-63767 (SaCas9) and EMD-63768 (eSaCas9-NNG). The NGS data have been deposited in the NCBI under accession code PRJNA1088532. All data are available in the manuscript or the supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJinek, M. \u003cem\u003eet al.\u003c/em\u003e A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e337\u003c/strong\u003e, 816\u0026ndash;821 (2012).\u003c/li\u003e\n\u003cli\u003eCong, L. \u003cem\u003eet al.\u003c/em\u003e Multiplex genome engineering using CRISPR/Cas systems. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e339\u003c/strong\u003e, 819\u0026ndash;823 (2013).\u003c/li\u003e\n\u003cli\u003eMali, P. \u003cem\u003eet al.\u003c/em\u003e RNA-guided human genome engineering via Cas9. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e339\u003c/strong\u003e, 823\u0026ndash;826 (2013).\u003c/li\u003e\n\u003cli\u003eSternberg, S. H., Redding, S., Jinek, M., Greene, E. C. \u0026amp; Doudna, J. A. 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However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Furthermore, the precise nuclease activation mechanism of SaCas9 remains elusive. Here, we rationally engineered a SaCas9 variant (eSaCas9-NNG) with an expanded target scope and reduced off-target activity. The eSaCas9-NNG induced indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice. We further determined the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for the improved specificity and illuminating notable differences in the activation mechanisms between the small SaCas9 and the larger SpCas9. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for \u003cem\u003ein vivo\u003c/em\u003e gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases.\u003c/p\u003e","manuscriptTitle":"Activation mechanism and molecular engineering of Staphylococcus aureus Cas9","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 07:22:38","doi":"10.21203/rs.3.rs-7614961/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5bedf02b-3edb-4238-a31d-48d00c4d5d42","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55033767,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":55033768,"name":"Biological sciences/Biotechnology/Gene therapy"}],"tags":[],"updatedAt":"2026-04-18T07:05:49+00:00","versionOfRecord":{"articleIdentity":"rs-7614961","link":"https://doi.org/10.1038/s41467-026-71626-2","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-04-16 04:00:00","publishedOnDateReadable":"April 16th, 2026"},"versionCreatedAt":"2025-09-22 07:22:38","video":"","vorDoi":"10.1038/s41467-026-71626-2","vorDoiUrl":"https://doi.org/10.1038/s41467-026-71626-2","workflowStages":[]},"version":"v1","identity":"rs-7614961","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7614961","identity":"rs-7614961","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}