Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2

Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2 | 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 Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2 Osamu Nureki, Satoshi Omura, Hayato Morinaga, Hisato Hirano, Yuzuru Itoh, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5481685/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jun, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ, a recently identified miniature type V effector encoded in phage genomes, has emerged as a promising candidate for genome editing due to its nuclease activity in mammalian and plant cells. However, the detailed molecular mechanisms of Casλ family of enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. The structures revealed that, unlike other type V CRISPR-Cas effectors, the REC2 domain directly interacts with the substrate DNA within the RuvC active site to facilitate the target DNA cleavage. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Molecular biology/CRISPR-Cas systems/CRISPR-Cas9 genome editing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems provide adaptive immunity against mobile genetic elements (MGEs) in prokaryotes and are divided into two classes (classes 1 and 2) and six types (types I–VI) 1,2 . Class 2 systems include types II, V, and VI, in which Cas9, Cas12, and Cas13, respectively, function as single, multidomain effectors to interfere with MGEs. Cas9 from Streptococcus pyogenes (SpCas9) associates with dual RNA guides (CRISPR RNA [crRNA] and trans-activating crRNA [tracrRNA]) and cleaves double-stranded DNA (dsDNA) targets at sequences complementary to a 20-nt guide segment in the RNA guide and flanked by NGG (where N is any nucleotide) protospacer adjacent motifs (PAMs), using its RuvC and HNH nuclease domains 3,4 . By contrast, Cas12a from Acidaminococcus sp. (AsCas12a) associates with a crRNA and cleaves dsDNA targets at sequences complementary to a 20-nt guide segment with TTTV (where V is A, G, or C) PAMs, using a single RuvC nuclease domain 5 . Since SpCas9 and AsCas12a exhibit robust DNA cleavage activities in eukaryotic cells, they are widely utilized as powerful genome engineering tools 5,6 . Although CRISPR-Cas systems are typically encoded in the genomes of bacteria and archaea, recent studies have described the widespread presence of diverse types of CRISPR-Cas systems encoded in bacteriophage genomes 7–11 . Among them, single multidomain CRISPR-Casλ effector proteins have been identified as compact RNA-guided DNA endonucleases 11 . The Mahaphage-encoded Casλ1 protein, consisting of 747 amino acid residues, is about half the size of SpCas9 and AsCas12a. Casλ1 binds to a crRNA and cleaves dsDNA targets with TTR (where R is A or G) PAMs, using its single RuvC nuclease domain 11 . Moreover, Casλ1 processes its cognate precursor crRNA (pre-crRNA) to the mature crRNA by using its RuvC domain, as observed in Cas12c and Cas12j (also known as CasΦ) 10–13 . Despite bearing little sequence similarity to known Cas12 proteins, these biochemical features of Casλ1 suggested its classification as a phage-encoded type V CRISPR-Cas effector 11 . However, the diversity and conservation of the biochemical properties among Casλ family enzymes are unclear, and the detailed molecular basis of DNA cleavage and guide RNA processing by Casλ remain enigmatic. Here, using metagenomic and phylogenetic analyses, we identified CRISPR-Casλ2 as a member of the Casλ family of enzymes. Biochemical analysis revealed the robust dsDNA cleavage activity of Casλ2, with an optimal 16–18-nt guide crRNA under physiological conditions, creating asymmetric staggered DNA double-stranded breaks distinct from the cleavage pattern observed with Casλ1. In addition, we showed that Casλ2 processes its pre-crRNA in a unique manner, in which Casλ2 functions as a “molecular ruler” to define the spacer lengths of the mature crRNA. We then determined the cryo-electron microscopy (cryo-EM) structures of Casλ2 in complex with the crRNA and dsDNA target in four distinct functional states, and with the pre-crRNA in one state, illustrating the stepwise domain rearrangements occurring during nuclease activation. Overall, this study advances our understanding of the molecular mechanisms of diverse type V CRISPR-Cas effectors and establishes a foundation for future engineering of Casλ enzymes. Results Characterization of the CRISPR-Casλ2 system A discovery campaign was previously initiated to identify diverse CRISPR-Cas systems, resulting in a database of candidate effectors 14,15 . From this database, we identified a putative type V Cas variant with an open reading frame (ORF) size of 741 amino acids and a unique RuvC domain within its C-terminal region. Metagenomic tracing confirmed that this effector was first isolated in a microbiome sample from Bos taurus , raised in Alberta, CA 16 . Phylogenetic analysis revealed that this newly identified variant clustered with a previously reported CRISPR-Casλ effector encoded by bacteriophage (Fig. 1 a). Based on this similarity, we designated this variant as CRISPR-Casλ2. To functionally characterize the CRISPR-Casλ2 system, we employed an Escherichia coli ( E. coli ) negative selection screen as described previously 14,15 . Briefly, we prepared a plasmid carrying the Casλ2 gene, a CRISPR array library targeting pACYC184 and essential E. coli genes, and concatenated cas gene-flanking noncoding sequences for the unbiased detection of a tracrRNA (Fig. 1 b). The plasmid was transformed into E. coli cells which were then grown under antibiotic selection. In screening strategy, when effector RNA-guided interference activity is present, bacterial viability is reduced. This reduction is detected by quantification of array depletion in the harvested cells, which revealed that Casλ2 demonstrated robust interference activity against dsDNA targets (Fig. 1 c). Similar activity was observed in both the presence and absence of the noncoding flanking sequences from the effector-encoding plasmid, indicating that Casλ2 efficiently targets dsDNA and functions with the cognate crRNA without the requirement of a tracrRNA. To identify the PAM specificity of Casλ2, we assessed the target-flanking sequences of the strongly depleted arrays in the E. coli screens. Deep sequencing analysis revealed that the dsDNA interference by Casλ2 depends on 5′-TTR-3′ (where R represents A or G) PAMs (Fig. 1 c). Subsequently, we investigated the crRNA-guided DNA cleavage activity of Casλ2 through in vitro DNA cleavage assays, using purified Casλ2, a crRNA with a 20-nt guide segment, and linearized plasmid DNA containing a target sequence and 5′-TTN-3′ PAMs. Casλ2 did not cleave target DNAs with 5′-TTY-3′ (where Y represents T or C) PAMs, but efficiently cleaved target DNAs with 5′-TTR-3′ PAMs, exhibiting slightly higher activity with the 5′-TTA-3′ PAM compared to the 5′-TTG-3′ PAM (Fig. 1 d). Collectively, these results indicate that CRISPR-Casλ2 is a variant of the phage-encoded CRISPR-Casλ family and exhibits crRNA-guided dsDNA cleavage activity with a 5′-TTR-3′ PAM. Biochemical characterization of Casλ2-mediated dsDNA cleavage To comprehensively assess the biochemical properties of Casλ2, we examined the in vitro DNA cleavage activity of purified Casλ2 using a linearized plasmid DNA target bearing a 5′-TTA-3′ PAM under various biochemical conditions. Casλ2 efficiently cleaved the target DNA over a temperature range of 37 to 55°C, with a preference for NaCl concentrations between 25 and 100 mM (Fig. 1 e and f). Casλ2 required spacer lengths of at least 16 nucleotides for effective dsDNA cleavage, with 16–18-nt guide crRNAs being optimal (Fig. 1 g). Casλ1 was previously reported to cleave the target DNA strand (TS) and non-target DNA strand (NTS) at 11–13 nt and 26–29 nt downstream of the PAM, respectively, generating cleavage products with pronounced staggered 5′-overhangs of 11–16 nt 11 . In contrast, Sanger sequencing of the target DNA cleavage products revealed that Casλ2 cleaved the TS at 23 nt downstream of the PAM and NTS at 19 or 20-nt downstream of the PAM, generating 3 or 4-nt 5′-overhangs (Fig. 1 h). Cryo-EM structure of the Casλ2–crRNA–target DNA ternary complex To understand the RNA-guided DNA cleavage mechanism of Casλ2, we determined the cryo-EM structure of Casλ2 in complex with a 56-nt crRNA and its 40-nt dsDNA target bearing the 5′-TTA-3′ PAM, at an overall resolution of 2.9 Å (Fig. 2 , Extended Data Fig. 1 , and Supplementary Tables S1 and S2). To prevent target DNA cleavage during the reconstruction, we used dsDNA with phosphorothioate modifications within the DNA backbone around the cleavage site. Casλ2 adopts a bilobed architecture consisting of recognition (REC) and nuclease (NUC) lobes, as observed in the structure of Casλ1 (Fig. 2 a–c). The REC lobe consists of the WED, REC1, and REC2 domains, while the NUC lobe has the RuvC, REC3, and target nucleic acid-binding (TNB) domains. The WED domain adopts a typical oligonucleotide/oligosaccharide-binding (OB) fold, consisting of seven β strands flanked by an α helix. The REC1 and REC2 domains comprise six α helices and four α helices, respectively, with each forming a characteristic globular shape. The RuvC domain has an RNaseH fold, consisting of a conserved five-stranded mixed β sheet flanked by four α helices, with Asp324, Glu526, and Asp680 forming an active center similar to those of other Cas12 enzymes (Extended Data Fig. 2 a and b) 13,17–22 . An α helix (referred to as the lid helix) is inserted between strand β4 and helix α3, corresponding to the region known as the lid motif in other Cas12 enzymes. Additionally, the RuvC domain of Casλ2 has unique insertion helices between the strands β2 and β3, and between the strand β3 and helix α1, expanding the size of the RuvC domain as compared with other Cas12 enzymes (Extended Data Fig. 2 a and b). The REC3 domain, which is not resolved in the previously reported Casλ1 structure, contains a two-stranded antiparallel β-sheet and two α helices, and is inserted between the strand β4 and lid helix of the RuvC domain (Fig. 2 b, c and Extended Data Fig. 2 b). A Dali search revealed that the REC3 domain of Casλ2 lacks structural similarity with any other known proteins, including those of Cas12 enzymes 23 . The TNB domain comprises a three-stranded mixed β-sheet and two α helices. The guide–target heteroduplex is accommodated within the central channel between the REC and NUC lobes, whereas the PAM-containing DNA duplex (PAM duplex) is sandwiched by the WED and REC1 domains (Fig. 2 b and c). The crRNA scaffold is bound within the groove formed by the WED and RuvC domains. Notably, the substrate single-stranded DNA (ssDNA) of the 3′ NTS is bound within the RuvC active site, which is not observed in the Casλ1 structure, indicating that the current structure represents a catalytically active state, as described below. crRNA architecture and recognition The crRNA consists of the 20-nt guide segment (G1 to C20) and the 36-nt repeat-derived scaffold (C(− 36) to C(− 1)) (Fig. 2 d and e). Notably, the crRNA scaffold comprises the stem and stem-loop, and lacks the pseudoknot structure conserved within most Cas12 enzymes (Extended Data Fig. 2 c) 13,17–20,24,25 . The stem contains a 5-bp duplex (G(− 33):C(− 1) to U(− 29):A(− 5)), while the stem-loop consists of a 6-bp distorted duplex (A(− 28):U(− 8) to U(− 20):A(− 14)) with a wobble base pair (U(− 23):G(− 12)) and two flipped-out bases (U(− 27) and U(− 22)), connected by a characteristic U-rich loop (U(− 19) to U(− 15)) (Fig. 2 d). The stem and stem-loop are connected with an approximate 80° bend, mediated by a two-adenine junction (A(− 7) and A(− 6)) (Fig. 2 e). The crRNA scaffold is recognized by Casλ2 through both base-specific and non-specific interactions (Fig. 3 and Extended Data Fig. 3 ). The stem is accommodated within the groove formed by the WED and RuvC domains, and primarily recognized by these domains through interactions with its sugar-phosphate backbone. In particular, the first G(− 33):C(− 1) base pair of the stem is recognized by Arg271 and Asn625 through hydrogen bonds, while the phosphate backbones of U(− 32) and G1, adjacent to the G(− 33):C(− 1) base pair, hydrogen bond with Lys8 and Tyr709, respectively (Fig. 3 b). The stem-loop is exposed to the solvent and has minimal interactions with the RuvC domain, with helix α5 inserted within the RuvC domain playing central roles in the stem-loop recognition. Specifically, Gln412, Lys413, and Lys416 form hydrogen bonds with the backbone phosphate group of U(− 27) (Fig. 3 c). Tyr409 stacks with U(− 27) and hydrogen bonds with the phosphate group of A(− 14) (Fig. 3 c). The U-rich loop is recognized by the RuvC domain through base-specific interactions. Specifically, U(− 17) is stabilized by coordination with the magnesium ion and forms base-specific hydrogen bonds with Lys436 and Asn495, suggesting that the U-rich loop plays an important role in the sequence-specific recognition of the crRNA by Casλ2 (Fig. 3 d). Collectively, these structural observations reveal how Casλ2 assembles with its cognate crRNA to form a ribonucleoprotein complex. Target DNA architecture and recognition The target DNA forms a 17-bp guide–target heteroduplex and 10-bp PAM duplex (Fig. 2 d). Nucleotides dG4* to dG18* and dT25* to dT30* in the NTS and dA(− 13) to dC(− 1) in the TS are not resolved in the density map, probably due to their flexibility. The guide–target heteroduplex is accommodated within the positively charged central groove formed by the REC1, REC2, REC3, and RuvC domains, and is recognized by these domains mainly through sugar-phosphate backbone interactions (Fig. 3 a and Extended Data Fig. 3 ). Lys2, which is not conserved in Casλ1, forms a hydrogen bond with the backbone phosphate group between dC17 and dT18 and a hydrophobic interaction with the first G1:dC17 base pair in the heteroduplex, suggesting that Casλ2 facilitates target DNA unwinding in a distinct manner from Casλ1 (Extended Data Figs. 3 and 4 ). Notably, Tyr548 in the REC3 domain stacks with the U17:dA1 base pair in the heteroduplex to preclude the extension of the heteroduplex beyond the protein, as observed in other Cas12 enzymes, explaining the 16–18-nt preference for Casλ2-mediated DNA cleavage (Fig. 3 e). In the present structure, the PAM duplex with the 5′-TTA-3′ PAM is bound between the WED and REC1 domains and recognized in a base-specific manner (Fig. 3 a and Extended Data Fig. 3 ). Specifically, the 5-methyl groups of dT(− 3*) and dT(− 2*) in the TTR PAM form hydrophobic interactions with Thr113 and Phe118, respectively (Fig. 3 f). The dA20 nucleobase, which base pairs with dT(− 3*), forms base-specific hydrogen bonds with Gln239 (Fig. 3 f). The N7 atom of dA(− 1*) hydrogen bonds with Asn89, explaining the requirement for the third R nucleotide in the TTR PAM (Fig. 3 f). In addition, the 5-methyl group of the dT18 nucleobase, which base pairs with dA(− 1*), forms hydrophobic interactions with Phe118 and Ile237, providing an explanation for the slight preference of Casλ2 for the TTA PAM rather than the TTG PAM (Fig. 3 f). Indeed, alanine substitutions of Asn89, Thr113, Phe118, Ile237, and Gln239 reduced or abolished the Casλ2-mediated DNA cleavage activity in vitro (Fig. 3 h). Taken together, these structural and biochemical observations reveal the mechanism of PAM recognition by Casλ2. Substrate DNA recognition within the RuvC active site In the Casλ2 structure, we observed a 6-nt ssDNA density bound within the groove between the RuvC and TNB domains, which is not observed in the Casλ1 structure (Extended Data Fig. 5 a). Nucleotides dG19* to dA24* in the NTS are well fitted in the density map and enter the RuvC catalytic site, indicating that the present structure likely represents the NTS-cleaving state (Fig. 3 g and Extended Data Fig. 5 a). The substrate NTS is recognized by Casλ2 in a sequence-independent manner. Notably, Tyr178 in the REC2 domain forms a hydrogen bond with the backbone phosphate of dG19*, while Pro721 in the TNB domain stacks with the base of dG19*, thereby anchoring dG19* at the entrance of the RuvC active site (Fig. 3 g). In addition, Phe533 in the RuvC domain inserts between dC20* and dA21* and forms stacking interactions with their nucleobases, kinking the substrate DNA around a scissile phosphate (Fig. 3 g). Tyr178 and Phe533 are well conserved in Casλ enzymes and their alanine substitutions reduced or abolished the in vitro DNA cleavage activities of Casλ2, confirming the importance of these residues for substrate DNA recognition (Fig. 3 h and Extended Data Fig. 4 ). The phosphate group of dA21* is bound to the RuvC active site via two magnesium ions (Mg A 2+ and Mg B 2+ , respectively), which are likely coordinated by Asp324, Glu526, and Asp680 (Fig. 3 g and Extended Data Fig. 5 b). Additionally, Asn326 in the β1 strand of the RuvC domain participates in the coordination of Mg A 2+ (Fig. 3 g and Extended Data Fig. 5 b). The alanine substitution of Asn326 almost completely eliminated the DNA cleavage activity, suggesting the important role of Asn326 for magnesium ion coordination besides the typical DED catalytic triad (Fig. 3 h). These structural and biochemical findings indicate that Casλ2 cleaves the target DNA in a manner dependent on two magnesium ions, as observed in other Cas12 enzymes. Concomitant conformational changes of Casλ2 for target DNA cleavage In addition to the NTS-cleaving state (State III), we found two distinct classes (States I and II) during the cryo-EM data analysis, and determined the two additional structures at overall resolutions of 2.9 (State I) and 3.1 (State II) Å (Extended Data Fig. 1 ). Although Casλ2 recognizes the target DNA and crRNA similarly in all three states, we observed notable structural differences in their REC2, RuvC, and TNB domains. In State I, Casλ2 adopts an open conformation, where the REC2 domain is distant from the NUC lobe with low local resolution, probably due to its flexibility (Fig. 4 a). Furthermore, in this state, part of the RuvC domain and the entire TNB domain (residues 633 to 741) are not resolved in the density map (Fig. 4 a and b). These structural observations suggest that this structure likely represents a “catalytically incompetent state”, in which the RuvC and TNB domains are flexible and the RuvC active site has not yet been formed. In contrast, in State II, the RuvC and TNB domains are fully resolved in the density map, while the REC2 domain remains distant from the NUC lobe (Fig. 4 c). In this state, although the RuvC active site is located at a position similar to that in the NTS-cleaving state, the substrate DNA strand is not bound within the RuvC domain (Fig. 4 d). Thus, this structure represents an “intermediate state”, in which the RuvC and TNB domains have become ordered but not yet adopted the proper local conformation for accommodating the substrate DNA strand within the groove between the RuvC and TNB domains. A structural comparison between the intermediate and NTS-cleaving states reveals the local conformational rearrangements of Casλ2 for accommodating the substrate DNA within the RuvC active site (Fig. 4 c–f and Extended Data Fig. 5 c–e). In the intermediate state, residues 527 to 540 (referred to as the lid loop here) are distant from the guide–target heteroduplex and occlude the RuvC active site (Extended Data Fig. 5 c). In the NTS-cleaving state, in contrast, Lys535 in the lid loop forms a hydrogen bond with the backbone phosphate of G11 in the heteroduplex, thereby facilitating the structural transition of the lid loop to open the RuvC active site (Extended Data Fig. 5 d). Additionally, we observed a concomitant conformational rearrangement between the REC2 and TNB domains. During the transition from the intermediate state to the NTS-cleaving state, the REC2 domain undergoes a significant movement of approximately 30 Å toward the TNB domain (Extended Data Fig. 5 e). This conformational change cooperatively induces the inward movement of a helix in the TNB domain toward the REC2 domain, providing interaction interfaces between the REC2 and TNB domains (Extended Data Fig. 5 e). Specifically, Tyr178 in the REC2 domain and Pro721 in the TNB domain form a stacking interaction, while Leu725 and His726 in the TNB domain form hydrophobic and hydrogen-bonding interactions with Tyr178 and Glu174 in the REC2 domain, respectively, thereby stabilizing the closed conformation of Casλ2 (Extended Data Fig. 5 d and e). This closed conformation enables Tyr178 and Pro721 to interact with the substrate DNA strand bound within the RuvC active site, as described above. Taken together, these structural observations demonstrate the concerted conformational changes of the REC2, RuvC, and TNB domains for target DNA cleavage by Casλ2. Target strand cleavage by Casλ2 Previous studies revealed that Cas12 enzymes cleave their dsDNA targets by using the single RuvC nuclease domain in a sequential manner, in which NTS cleavage is followed by TS cleavage 26–28 . To elucidate the TS cleavage mechanism of Casλ2, we used a 14-nt NTS and a 40-nt TS during complex reconstruction to mimic the post-NTS-cleavage state, and determined the ternary complex structure at an overall resolution of 2.8 Å (Extended Data Fig. 6 and Supplementary Table S1 ). Although the overall structure is similar to the NTS-cleaving state, we observed the extended TS density entering the RuvC active site, suggesting that this structure represents the TS-cleaving state (Fig. 4 g). The extended TS is kinked at the end of the guide–target heteroduplex and folded back along the REC3 domain to access the RuvC domain (Fig. 4 g). The single-stranded segment of the TS beyond the guide–target heteroduplex is recognized by Casλ2 in an almost identical manner to that observed in the NTS-cleaving state. Tyr178 and Pro721 form hydrogen-bonding and stacking interactions with dT(− 7), respectively (Fig. 4 h). Phe533 forms stacking interactions with dG(− 6) and dG(− 5), kinking the TS around the scissile phosphate (Fig. 4 h). The phosphate group of dG(− 5), located 23 nt downstream from the PAM, is bound to the RuvC active site with two magnesium ions, coordinated by Asp324, Asn326, Glu526, and Asp680, explaining the TS cleavage position 23-nt downstream from the PAM observed in our biochemical analysis (Figs. 1 h and 4 h). Together, these structural observations indicate that Casλ2 cleaves both the NTS and TS in an identical manner within the RuvC active site, with the REC3 domain providing a pathway for the TS toward the RuvC active site. Guide RNA processing by the Casλ2 Recent studies have shown that Casλ1 processes its pre-crRNA at the spacer region by using the RuvC active site 11 . To determine whether Casλ2 is also capable of processing its pre-crRNA into the mature crRNA, we performed an in vitro pre-crRNA processing assay using two types of pre-crRNAs, composed of a 5′-spacer–repeat–spacer-3′ (srs) or 5′-repeat–spacer–repeat-3′ (rsr) sequence (Fig. 5 a). The rsr-type pre-crRNA was successfully processed by Casλ2, while the srs-type pre-crRNA was not (Fig. 5 b). We also found that the D324A mutant (dCasλ2) lost the pre-crRNA processing activity (Fig. 5 b). Together, these results indicate that Casλ2 cleaves the 3′ side of the pre-crRNA by using the RuvC active site, as observed in Casλ1. In addition to the Casλ enzymes, Cas12c and Cas12j also process their pre-crRNAs at their RuvC active sites through distinct mechanisms 10,13 . Cas12c recognizes both the upstream and downstream repeat sequences of the pre-crRNA and cleaves immediately upstream of the downstream repeat sequence, whereas Cas12j cleaves within the upstream spacer region of the pre-crRNA. To clarify the pre-crRNA processing mechanism by Casλ2 in detail, we prepared the rsr-type pre-crRNA with different lengths of spacers (17 nt and 24 nt, respectively) and examined the processing patterns of Casλ2 against these pre-crRNAs (Fig. 5 c). Casλ2 successfully cleaved both pre-crRNAs. Notably, while the lengths of the cleaved repeat–spacer segments were not changed, the lengths of the cleaved downstream repeats changed depending on the spacer length of the pre-crRNA (Fig. 5 d). We also conducted in vitro processing assays using a pre-crRNA-polyA substrate, in which the downstream repeat was replaced with a 20-nt polyA sequence, and observed similar processing patterns (Fig. 5 d). These results indicated that, unlike Cas12c and Cas12j, Casλ2 recognizes the upstream repeat sequence and cleaves the spacer region by a unique ruler mechanism, in which Casλ2 functions as a “molecular ruler” to define the spacer lengths of the mature crRNA. Next, to examine the spacer length of the mature crRNA defined by Casλ2, we compared the lengths of the cleaved repeat–spacer products with those of synthesized crRNAs with different spacer lengths. A denaturing urea polyacrylamide gel electrophoresis analysis revealed that the spacer length of the mature crRNA varies between 16–18 nt, which is consistent with the optimal spacer length of approximately 16–18 nt for target DNA cleavage by Casλ2 (Fig. 5 e). Collectively, our biochemical data indicate that Casλ2 processes its pre-crRNA through a ruler mechanism, in which Casλ2 recognizes the upstream repeat sequence and cleaves downstream of the recognition site at a defined length of 16–18 nt. Cryo-EM structure of the Casλ2–crRNA binary complex To obtain mechanistic insights into the pre-crRNA processing by the Casλ2 ruler mechanism, we determined the cryo-EM structure of Casλ2 (the inactive D324A mutant) in complex with a pre-crRNA-polyA (76 nt) at an overall resolution of 3.0 Å (Figs. 5 c, f, Extended Data Fig. 7 a–f, and Supplementary Table S1 ). In the binary complex structures of most Cas12 enzymes, a 5–7 nt spacer is ordered in an A-form geometry as a seed 13,18,21,29 . In contrast, the binary complex structure of Casλ2 revealed that the relatively long 12-nt spacer (G1 to G12) is ordered within the positively charged central groove in a stretched conformation, while nucleotides C13 to A40 are disordered probably due to their flexibility (Fig. 5 g). The 12-nt spacer is extensively recognized by Casλ2 in a sequence-independent manner. Specifically, Tyr709, His230, Phe222, Tyr448, and Tyr548 form stacking interactions with G1, A5, U6, A8, and U11, respectively, thereby anchoring the 12-nt spacer in a stretched conformation rather than an A-form geometry (Fig. 5 g). These structural observations suggest that Casλ2 protects a 12-nt spacer from enzymatic cleavage by accommodating it within its central groove. Subsequently, the remaining downstream spacer is bent and spontaneously directed toward the RuvC active site, resulting in pre-crRNA cleavage at a position 16–18 nt downstream from the crRNA scaffold (Fig. 5 h). A structural comparison between the binary and ternary (NTS-cleaving state) complexes revealed structural rearrangements in the REC1 and REC2 domains upon target DNA binding (Extended Data Fig. 7 g–i). The ternary complex adopts an open conformation, in which the REC1 domain is distant from the RuvC domain to form a binding groove for the guide–target heteroduplex. In contrast, the binary complex adopts a closed conformation, ensuring mutual interactions between the protein and 12-nt spacer. Notably, Phe222 and His230 undergo 5-Å and 14-Å shifts downward toward the RuvC domain, respectively, to form stacking interactions with the spacer (Extended Data Fig. 7 g–i). In addition, in the binary complex, the REC2 domain interacts with the TNB domain, as also observed in the NTS-cleaving state, suggesting that the binary complex is already prepared to accommodate the single-stranded RNA within the RuvC active site for pre-crRNA processing. Together, these structural observations illustrate how Casλ2 assembles with the pre-crRNA prior to the target DNA binding and functions as a molecular ruler to define the spacer length of the mature crRNA. Discussion In this study, we identified Casλ2 through a metagenomic analysis as a compact CRISPR-Cas effector belonging to the same branch as Casλ1, a type V CRISPR-Cas effector identified in phage genomes. Our biochemical data demonstrated that Casλ2 exhibits both crRNA-guided DNA cleavage and pre-crRNA processing activities, using a single RuvC active site. Notably, by accommodating a relatively long 12-nt spacer within the central groove, Casλ2 functions as a molecular ruler to define the spacer lengths of the mature crRNA. Furthermore, we determined the cryo-EM structures of Casλ2 in five distinct states, shedding light on the activation mechanism of Casλ enzymes. A recent structural study revealed the crRNA-guided DNA binding mechanism of Casλ1 11 . However, the structure represents the inactive state wherein the substrate DNA is not bound within the RuvC active site, and thus the activation mechanism of Casλ family enzymes remained elusive. The cryo-EM structures of the Casλ2–crRNA binary complex and the Casλ2–crRNA–target DNA ternary complexes in four different functional states illuminated significant conformational rearrangements during the nuclease activation of Casλ2 (Fig. 6 and Supplementary Movie S1). In the binary complex, Casλ2 adopts a closed conformation, accommodating a relatively long 12-nt spacer within the central groove. Upon target DNA binding after pre-crRNA processing, Casλ2 transitions to an open conformation to accommodate the guide–target heteroduplex, with the REC2 domain dissociating from the TNB domain (incompetent state). In this incompetent state, the RuvC active site is not formed due to disorder in part of the RuvC domain and the entire TNB domain. Subsequently, the RuvC and TNB domains become ordered, accompanying the formation of the RuvC active site (intermediate state). In the intermediate state, the REC2 domain remains distant from the TNB domain, and the substrate DNA is not yet bound to the RuvC active site. Then, a substantial movement of the REC2 domain toward the TNB domain, accompanied by a local conformational rearrangement of the TNB domain, enables the NTS to enter the RuvC active site (NTS-cleaving state). Cleavage of the NTS is followed by the TS being directed along the positively charged surface of the REC3 domain to the RuvC active site (TS-cleaving state). These structural observations underscore the essential domain movements and conformational rearrangements in the activation of Casλ2. A structural comparison of Casλ2 with Casλ1 highlighted the mechanistic conservation and differences among the Casλ family enzymes (Fig. 7 ). Although Casλ1 and Casλ2 share limited sequence similarity (37% identity), their protein structures show a high degree of structural homology, with a root mean square deviation of 0.912 Å across 476 pruned atom pairs (Fig. 7 and Extended Data Fig. 4 ). The crRNA scaffold adopts almost identical configurations in both complexes, being accommodated within the groove between WED and RuvC domains in similar manners. The PAM duplex is commonly bound between the WED and REC1 domains. In contrast to these conserved structural features, there is a notable structural difference in PAM-distal guide–target heteroduplex recognition. Casλ2 recognizes a 17-bp guide–target heteroduplex, with the RuvC-inserted REC3 domain playing pivotal role in distinguishing the 17th base pair. In contrast, the REC3 domain in the Casλ1 structure is disordered, accommodating a shorter 16-bp guide–target heteroduplex within the central channel (Fig. 7 ). Although further biochemical characterization is needed, these structural observations suggest that Casλ2 identifies the target DNA more strictly than Casλ1 by recognizing the end of the heteroduplex through the REC3 domain. Structural comparisons of Casλ2 with CasΦ enhanced our understanding of the conserved structural features among the phage-encoded type V CRISPR-Cas effectors (Fig. 7 ). Despite their limited sequence similarity, both enzymes have similar sizes (CasΦ: 757 amino acids, Casλ2: 741 amino acids) and commonly adopt a bilobed architecture consisting of the REC and NUC lobes, with the guide–target heteroduplex accommodated within the central channel. Both enzymes do not require tracrRNAs and rely solely on crRNAs, which adopt a simple stem-loop configuration lacking a pseudoknot structure. Recent studies suggested that, during their evolution from TnpB, Cas12 enzymes acquired the ability to recognize the PAM-distal region of the guide–target heteroduplex through the insertion of the REC2 domain between their REC1 and WED domains (as seen in Cas12a), or through dimerization 25,30 (Fig. 7 ). Consistent with this hypothesis, CasΦ and Casλ2 recognize longer 18- and 17-bp guide–target heteroduplexes, respectively, compared to the 12-bp heteroduplex observed in the TnpB structure. Intriguingly, unlike most Cas12 enzymes, CasΦ and Casλ2 recognize the PAM-distal region of the guide–target heteroduplex through the inserted region within the RuvC domain (REC3 domain in Casλ2), rather than through the REC2 domain (Fig. 7 ). Instead, the REC2 domain of Casλ2 recognizes the substrate DNA within the RuvC active site. These findings imply that the phage-encoded type V CRISPR-Cas effectors have evolved from TnpB independently of those encoded by bacteria or archaea. In summary, our biochemical and structural analyses of Casλ2 advance our comprehension of the diverse phage-encoded type V CRISPR-Cas effectors. Phage genomes reportedly serve as natural reservoirs of miniature CRISPR-Cas effectors, which hold great promise as in vivo genome editing platforms using a cargo-size-limited, single AAV vector. Therefore, our structural findings provide the molecular framework for the development of compact genome editing technologies for in vivo gene therapy. Declarations Acknowledgements Arbor Biotechnologies is a privately funded company. 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. performed biochemical and structural analyses with help from H.M.; S.N.O., H.H., and Y.I. built the models and performed structural refinement; D.R.C. and D.A.S. designed and implemented the computational search; D.A.S., W.X.Y., and L.E.A. designed the E. coli negative selection screen; L.E.A., A.O., G.M., and A.J.G. performed experimental screens and validation; S.N.O., L.E.A., Z.M. and O.N. wrote the manuscript with help from all authors; and T.D., G.R.H., Z.M., and O.N. supervised the research. Declaration of interests G.M., A.J.G., A.O., L.E.A., G.R.H., T.D., W.X.Y., D.R.C., D.A.S., and Z.M. are current or former employees and shareholders of Arbor Biotechnologies. Arbor Biotechnologies has filed patents 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 Casλ2–crRNA–target DNA ternary complexes have been deposited in the Protein Data Bank under the accession codes 9IZP (incompetent state), 9IZQ (intermediate state), 9IZR (NTS-cleaving state), and 9IZS (TS-cleaving state). The atomic model of the Casλ2–crRNA binary complex has been deposited in the Protein Data Bank under the accession code 9IZM. The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-61038 (incompetent state), EMD-61039 (intermediate state), EMD-61040 (NTS-cleaving state), EMD-61041 (TS-cleaving state), and EMD-61037 (binary complex). RESOURCE AVAILABILITY 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] ). Material availability All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement. Data and code availability The atomic models of Casλ2–crRNA–target DNA ternary complexes have been deposited in the Protein Data Bank under the accession codes 9IZP (incompetent state), 9IZQ (intermediate state), 9IZR (NTS-cleaving state), and 9IZS (TS-cleaving state). The atomic model of the Casλ2–crRNA binary complex has been deposited in the Protein Data Bank under the accession code 9IZM. The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-61038 (incompetent state), EMD-61039 (intermediate state), EMD-61040 (NTS-cleaving state), EMD-61041 TS-cleaving state), and EMD-61037 (binary complex). This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the Lead Contact upon request. METHOD DETAILS E. coli negative selection screen Metagenomic data mining of run accession SRR5678926, using previously described computational discovery processes, identified an ORF associated with a CRISPR array. These elements were probed for activity using an enhanced version of the previously described E. coli negative selection screen 14,15 , wherein RNA-programmable CRISPR-Cas interference yields spacer-specific loss of antibiotic resistance or self-targeting of essential genes, resulting in cell death. Briefly, the E. coli codon-optimized genes representing the minimal CRISPR effector and accessory proteins +/- noncoding sequences were synthesized (GenScript) and cloned into a vector derived from pET-28a(+) (EMD-Millipore). The vector included an acceptor site for a CRISPR array library under the expression of the J23119 promoter. The CRISPR array library was composed of the “repeat-spacer-repeat” architecture, where “repeat’’ represents the consensus direct repeat sequence found in the CRISPR array associated with the effector, and ‘‘spacer’’ represents a variable sequence with a length determined by the mode of the spacer lengths found in the endogenous CRISPR array. The CRISPR array library containing 8,900 spacers, targeting either select E. coli essential genes or regions of pACYC184, was synthesized (Twist), amplified to introduce cloning handles and unique molecular identifiers (UMIs), and then bidirectionally cloned into the library acceptor site using Golden Gate assembly. The assembled plasmid library was electroporated into custom E. coli EXPRESS BL21 (DE3) electrocompetent cells (Lucigen) harboring the pACYC184 plasmid, encapsulated in droplets using a microfluidic device manufactured in-house, and grown under antibiotic selection at 37°C with shaking. After approximately 16h, the cells were isolated from the droplets, plasmid DNA was extracted using a QIAprep Spin Miniprep kit (Qiagen), and total RNA was extracted using a custom procedure based on the Agencourt RNAdvance Tissue Kit (Beckman Coulter). PCR was conducted on the plasmid DNA library (pre-transformation and post-harvest) using custom primers that amplify the CRISPR array and simultaneously introduce barcodes and sequencing adapters compatible with Illumina chemistry. The amplicons were pooled and then sequenced on a NextSeq 550 sequencer (Illumina). The NGS data were analyzed as described previously to determine interference activity, tracrRNA requirement, and PAM specificity 14,15 . Protein and RNA preparation for biochemical and structural analyses The gene encoding full-length Casλ2 (residues 1–741) was codon optimized, synthesized (Eurofins Genomics), and cloned into a modified pE-SUMO vector (LifeSensors). The mutations were introduced by a PCR-based method, using the vector encoding full-length Casλ2 as the template, and the sequences were confirmed by DNA sequencing (Supplementary Table S3). The Casλ2 proteins were expressed and purified using the protocol reported previously 13,20,25 . Briefly, the N-terminally His 6 -tagged Casλ2 proteins were expressed in Escherichia coli Rosetta2 (DE3). Transformed E. coli cells were cultured at 37°C until the OD 600 reached 0.8, and protein expression was then induced by the addition of 0.1 mM isopropyl β-D-thiogalactopyranoside (Nacalai Tesque). E. coli cells were further cultured at 20°C overnight and harvested by centrifugation. The cells were then resuspended in buffer A (20 mM HEPES-NaOH, pH 7.6, 20 mM imidazole, and 1 M NaCl), lysed by sonication, and centrifuged. The supernatant was mixed with 3 ml Ni-NTA Superflow resin (QIAGEN), and the mixture was loaded into an Econo-Column (Bio-Rad). Proteins were eluted with buffer B (20 mM HEPES-NaOH, pH 7.6, 0.3 M imidazole, 0.3 M NaCl) and then loaded onto a 5-ml HiTrap Heparin HP column (GE Healthcare) equilibrated with buffer C (20 mM HEPES-NaOH, pH 7.6, and 0.3 M NaCl). The proteins were eluted with a linear gradient of 0.5–2 M NaCl. The purified proteins were stored at −80°C until use. The crRNAs for structural analysis were transcribed in vitro with T7 RNA polymerase and purified by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis. The crRNAs for biochemical analysis were synthesized (Ajinomoto Bio-Pharma). In vitro DNA cleavage experiments The DNA cleavage activity of Casλ2 was measured by in vitro DNA cleavage experiments. First, these experiments were performed with the purified Casλ2, the crRNA containing a 20-nt spacer, and linearized pUC119 plasmids (100 ng, 4.7 nM) containing the target sequence with the TTN PAM. Next, the cleavage activities of Casλ2 were measured, using the linearized pUC119 plasmid with the TTA PAM. The linearized pUC119 plasmid (100 ng, 4.7 nM) was incubated at 37°C (25–75°C for Fig. 1g) for 5–60 min with the Casλ2–crRNA complex (750 nM) in 20 μL of reaction buffer, containing 20 mM HEPES-NaOH, pH 7.6, 50 mM NaCl (25–200 mM NaCl for Fig. 1f), 10 mM MgCl 2 , 1 mM DTT, and 2% 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). Electron microscopy sample preparation and data collection The Casλ2–crRNA–target DNA ternary complex was reconstituted by mixing purified Casλ2, the 56-nt crRNA, the 40-nt target DNA, and the 40-nucleotide non-target DNA at a molar ratio of 1:1.2:1.4:1.4. Each DNA strand has phosphorothioate modifications within the phosphate backbone around the cleavage site to inhibit DNA hydrolysis. The Casλ2–crRNA–target DNA ternary complex in the TS-cleaving state was reconstituted by mixing purified Casλ2, the 56-nt crRNA, the 40-nt target DNA, and the 14-nucleotide non-target DNA at a molar ratio of 1:1.2:1.4:1.4, with the target DNA strand bearing phosphorothioate modifications within the phosphate backbone around the cleavage site. The Casλ2 (D324A)–pre-crRNA binary complex was reconstituted by mixing purified Casλ2 and the 76-nt pre-crRNA at a molar ratio of 1:1.2. The Casλ2–crRNA–target DNA ternary complexes and the Casλ2 (D324A)–pre-crRNA binary complex were purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated with buffer D (20 mM HEPES-NaOH, pH 7.6, 50 mM NaCl, 10 mM MgCl 2 , 10 μM ZnCl 2 , and 1 mM DTT). The purified complex solutions (A 260 nm = 3 for the ternary complexes and A 260 nm = 2 for the binary complex) were then applied to Au 300-mesh R1.2/1.3 grids (Quantifoil) that were glow-discharged after adding 3 μL amylamine 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 of the Casλ2–crRNA–target DNA ternary complexes were collected with a total dose of approximately 50 electrons per Å 2 per 48 frames by the standard mode, and datasets of the Casλ2–pre-crRNA binary complex were collected with a total dose of approximately 50 electrons per Å 2 per 64 frames by the CDS mode, using the EPU software (Thermo Fisher Scientific). The dose-fractionated movies were subjected to beam-induced motion correction and dose weighting using MotionCor2 implemented in RELION-3.1, and the contrast transfer function (CTF) parameters were estimated using Patch-based CTF estimation in cryoSPARC v3.3.2 31,32 . Single-particle cryo-EM data processing Data were processed using cryoSPARC v3.3.2 and RELION-3.1 32,33 . For the Casλ2–crRNA–target DNA ternary complex, 4,084,635 particles were initially picked from the 3,087 motion-corrected and dose-weighted micrographs using Template picker, and extracted at a pixel size of 3.32 Å in the cryoSPARC platform. These particles were subjected to several rounds of cryoSPARC 2D classification to curate particle sets. The particles were further curated by heterogeneous refinement, using maps derived from ab initio reconstruction as templates. The selected particles were subjected to 3D variability analysis and the maps with different conformations were used for subsequent heterogeneous refinement, resulting in the reconstructions of three distinct conformational states (State I, the catalytically incompetent state; State II, the intermediate state; State III, the NTS-cleaving state) 34 . Particles corresponding to each state were imported into RELION and subjected to 3D classification without alignment. The particle sets with the most detailed features after 3D classification were subjected to Bayesian polishing 35 . The resulting particles were then imported back to cryoSPARC, and Non-uniform refinement yielded maps at 2.89 Å (State I, the catalytically incompetent state), 3.06 Å (State II, the intermediate state), and 2.93 Å (State III, the NTS-cleaving state) resolutions, according to the Fourier shell correlation (FSC) criterion of 0.143 36,37 . The local resolution was estimated by BlocRes in cryoSPARC. The datasets for the Casλ2–crRNA–target DNA ternary complex in the TS-cleaving state and the Casλ2–pre-crRNA binary complex were processed using cryoSPARC and RELION in similar manners. For data processing details, see Extended Data Fig. 1 (the catalytically incompetent state, the intermediate state, and the NTS-cleaving state), 6 (the TS-cleaving state), and 7 (the binary complex). Model building and validation The models were built using the protein model derived from the SWISS-MODEL server as the reference, followed by manual model building with Coot 38–42 . The models were refined using phenix.real_space_refine version 1.20.1, with secondary structure and metal coordination restraints 43 . The metal coordination restraints were generated using ReadySet, as implemented in PHENIX. The stereochemical restraints for phosphorothioate modified DNA links were generated manually. The models were validated using MolProbity 44 . Molecular graphics figures were prepared with UCSF ChimeraX-1.7.1 45 . In vitro pre-crRNA processing experiments The pre-crRNAs were transcribed in vitro with T7 RNA polymerase and purified by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis. The purified Casλ2 proteins and the pre-crRNAs were mixed at a molar ratio of 2:1 in 20 μL of reaction buffer, containing 20 mM HEPES-NaOH, pH 7.6, 50 mM NaCl, 10 mM MgCl 2 , 1 mM DTT, and 2% glycerol, and then incubated at 37°C for 30 min. The reactions were stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng), and then analyzed by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis. The gels were stained with SYBR Gold (Invitrogen). In vitro processing experiments were performed at least three times. QUANTIFICATION AND STATISTICAL ANALYSIS All data are expressed as mean ± SD. No statistical methods were used to predetermine sample size. Sample size was based on experimental feasibility and sample availability. 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Protein Sci. 30 , 70–82 (2021). Additional Declarations Yes there is potential Competing Interest. G.M., A.J.G., A.O., L.E.A., G.R.H., T.D., W.X.Y., D.R.C., D.A.S., and Z.M. are current or former employees and shareholders of Arbor Biotechnologies. Arbor Biotechnologies has filed patents related to this work. O.N. is a co-founder, board member, and scientific advisor of Curreio. The remaining authors declare no competing interests. Supplementary Files Supplementarytables.pdf Supplementary Tables S1-S2 CasLambda2NatCommunExtendedFig.pdf Supplementary Figure Legends Extended Data Fig. 1 | Single-particle cryo-EM analysis of the Casλ2–crRNA–target DNA ternary complex. (a) Size-exclusion chromatography profile of the Casλ2–crRNA–target DNA complex. The peak fraction indicated as the complex was used for the following cryo-EM analyses. (b) Representative cryo-EM micrograph, recorded on a 300-kV Titan Krios microscope with a K3 camera. (c) Representative 2D class average images. (d) Single-particle cryo-EM image processing workflow. The data processing done in cryoSPARC is shown in blue, while the processing done in RELION is shown in black. (e–g) Cryo-EM density maps of the Casλ2–crRNA–target DNA complexes in the catalytically incompetent (e), intermediate (f), and NTS-cleaving (g) states, according to the local resolution. (h–j) Fourier shell correlation curves for the 3D reconstructions of the Casλ2–crRNA–target DNA complexes in the catalytically incompetent (h), intermediate (i), and NTS-cleaving (j) states. Extended Data Fig. 2 | RuvC domain and crRNA structures. (a) The RuvC domain structure of Casλ2 and that of UnCas12f (PDB: 7C7L). The RuvC domains comprise the RNaseH fold. The conserved β strands and α helices are numbered in blue and red, respectively, while the additional helices observed in Casλ2 are numbered in green. The conserved RuvC active site is indicated as red spheres. (b) Schematic of the RuvC domain of Casλ2. (c) Structures of crRNAs of Casλ2 (left), Cas12m2 (center) (PDB: 8HHL), and Cas12a (right) (PDB: 6I1K). While the crRNAs of most of the Cas12 enzymes, including Cas12m2 and Cas12a, comprise the conserved pseudoknot structure, the crRNA of Casλ2 comprises the stem and stem-loop, but lacks the pseudoknot structure. PK, pseudoknot. Extended Data Fig. 3 | Schematic of nucleic acid recognition. Residues that interact with nucleic acids through their main chains are shown in parentheses. The disordered regions are indicated by dashed gray lines. Extended Data Fig. 4 | Multiple sequence alignment of Casλ orthologues. Multiple sequence alignment of Casλ1, Casλ2, and DAE37039 (a hypothetical protein from Bacteriophage sp. that demonstrated sequence similarity to Casλ2 in a BLAST analysis). The secondary structure of Casλ2 is indicated above the sequences. The additional helices observed in the RuvC domain of Casλ2 are numbered in green, as in Figure S2A. The key residues of Casλ2 are marked with triangles below the sequences. The figure was prepared using Clustal Omega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo) and ESPript3 (https://espript.ibcp.fr/ESPript/ESPript/). Extended Data Fig. 5 | Map-model superimposition and conformational changes of Casλ2. (a) Overall cryo-EM density map (left) and map-model superimposition of the substrate NTS bound within the RuvC active site (right). (b) Close-up views of the RuvC active site of Casλ2 in the NTS-cleaving state. Cryo-EM density maps are shown as translucent gray surfaces. The bound Mg 2+ ions are shown as green spheres. (c and d) Structures (top) and close-up views around the RuvC domain (bottom) of the Casλ2–crRNA–target DNA complexes in the intermediate (C) and NTS-cleaving (D) states. Hydrogen bonds are shown as dashed lines. (e) Superimposition of the intermediate (orange) and NTS-cleaving (colored as in D) states. The top and bottom panels display the overall structure and a close-up view around the RuvC active site, respectively. The inward movement of the REC2 domain toward the TNB domain is accompanied by the inward movement of a helix in the TNB domain toward the REC2 domain. The substrate NTS in the NTS-cleaving state clashes with the lid loop in the intermediate state. Extended Data Fig. 6 | Single-particle cryo-EM analysis of the Casλ2–crRNA–target DNA ternary complex in the TS-cleaving state. (a) Schematic of the target DNA construct used in the complex reconstruction. Phosphorothioate modified regions in the target DNA strand are shown in green. In this panel, please change phosphorthioate to phosphorothioate. (b) Size-exclusion chromatography profile of the Casλ2–crRNA–target DNA complex in the TS-cleaving state. The peak fraction indicated as the complex was used for the following cryo-EM analyses. (c) Representative cryo-EM micrograph, recorded on a 300-kV Titan Krios microscope with a K3 camera. (d) Single-particle cryo-EM image processing workflow. The data processing done in cryoSPARC is shown in blue, while the processing done in RELION is shown in black. (e) Fourier shell correlation curve for the 3D reconstruction. (f) Cryo-EM density map according to the local resolution. Extended Data Fig. 7 | Single-particle cryo-EM analysis of the dCasλ2–pre-crRNA binary complex. (a) Size-exclusion chromatography profile of the dCasλ2–pre-crRNA complex. The peak fraction indicated as the complex was used for the following cryo-EM analyses. (b) Representative cryo-EM micrograph, recorded on a 300-kV Titan Krios microscope with a K3 camera. (c) Representative 2D class average images. (d) Single-particle cryo-EM image processing workflow. The data processing done in cryoSPARC is shown in blue, while the processing done in RELION is shown in black. (e) Fourier shell correlation curve for the 3D reconstruction. (f) Cryo-EM density map according to the local resolution. (g and h) Structures (top) and close-up views (bottom) of the Casλ2–crRNA–target DNA complex in the NTS-cleaving state (g) and the dCasλ2–pre-crRNA binary complex (h). (i) Superimposition of the NTS-cleaving state (orange) and the binary complex (colored as in h). The top and bottom panels display the overall structure and a close-up view, respectively. SupplementaryMovieS1.mp4 Supplementary Movie Legend Supplementary Movie S1 | Conformational rearrangements of Casλ2 during its nuclease activation. Stepwise domain rearrangements of Casλ2. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5481685","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":385891482,"identity":"d6e24e67-22a9-42b5-9aa2-410cf1030426","order_by":0,"name":"Osamu 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Biotechnologies","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Scott","suffix":""},{"id":385891496,"identity":"e570ad1f-8e43-4225-be1d-1ed43a08a020","order_by":14,"name":"Zachary Maben","email":"","orcid":"","institution":"Arbor Biotechnologies","correspondingAuthor":false,"prefix":"","firstName":"Zachary","middleName":"","lastName":"Maben","suffix":""}],"badges":[],"createdAt":"2024-11-19 08:40:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5481685/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5481685/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-08300-8","type":"published","date":"2025-06-05T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78825554,"identity":"4a0ea24c-2d10-4efb-9a1a-f4e4c24fe9ba","added_by":"auto","created_at":"2025-03-19 12:23:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":558407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiscovery and biochemical characterization of Casλ2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) CRISPR-Cas12 effectors exhibit significant diversity, with Casλ1 and Casλ2 clustering together alongside other representative type V Cas systems. Protein sequences were classified through global alignment using free end gaps and similarity scoring based on the Blosum80 matrix.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Schematic of the \u003cem\u003ein vivo\u003c/em\u003e \u003cem\u003eE. coli\u003c/em\u003e negative selection screen.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Depletion activity of effector using a pooled depletion screen. Points represent UMIs corresponding to a specific spacer sequence. The depletion score for spacer sequences was measured as the minimum score among replicates of the ratio between normalized input reads to normalized output reads. (left) Sequence logos of spacer sequences with depletion scores greater than 3.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of Casλ2 with 20-nt guide crRNA. The linearized plasmid targets bearing the TTN PAMs were incubated with the Casλ2–crRNA complex at 37°C for 30 min. The cleavage products were then analyzed by a MultiNA microchip electrophoresis system.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of Casλ2 with 20-nt guide crRNA-temperature optimization. The linearized plasmid target bearing the TTA PAM was incubated with the Casλ2–crRNA complex at 25, 37, 45, 55, and 65°C for 5 and 30 min, in reaction buffer containing 50 mM NaCl. 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\u003ef\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of Casλ2 with 20-nt guide crRNA-NaCl concentration optimization. The linearized plasmid target bearing the TTA PAM was incubated with the Casλ2–crRNA complex at 37°C for 5 and 30 min, in reaction buffers with NaCl concentrations varying between 25, 50, 100, and 200 mM. 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\u003eg\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of Casλ2 with 12–22-nt guide crRNAs. The linearized plasmid target bearing the TTA PAM was incubated with the Casλ2–crRNA complex at 37°C for 5 and 30 min, in reaction buffer containing 50 mM NaCl. 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\u003eh\u003c/strong\u003e) Cleavage sites in the target DNA.\u003c/p\u003e","description":"","filename":"CasLambda2NatCommunMainFig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/4f8a95b92ebe96746fdb53ad.jpg"},{"id":78825551,"identity":"d406be80-9c10-439e-bd42-8fee56733497","added_by":"auto","created_at":"2025-03-19 12:23:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":754407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structure of the Casλ2–crRNA–target DNA complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Domain structure of Casλ2.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb \u003c/strong\u003eand\u003cstrong\u003e c\u003c/strong\u003e) Cryo-EM density maps (\u003cstrong\u003eb\u003c/strong\u003e) and ribbon models (\u003cstrong\u003ec\u003c/strong\u003e) of the Casλ2–crRNA–target DNA complex. Magnesium ions are shown as green spheres. The disordered NTS is indicated by dashed lines. TS, target DNA strand; NTS, non-target DNA strand.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Schematic of the crRNA and target DNA. The disordered regions are enclosed by dashed boxes.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Structure of the crRNA.\u003c/p\u003e","description":"","filename":"CasLambda2NatCommunMainFig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/74ad9240fd4bcc42ec3de457.jpg"},{"id":78825552,"identity":"3c92aca4-84ec-4dec-b516-aaae27136e21","added_by":"auto","created_at":"2025-03-19 12:23:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":888368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecognition of the crRNA and target DNA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Recognition of the crRNA and target DNA by Casλ2. The Casλ2 proteins are shown as surface models.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb–d\u003c/strong\u003e) Recognition of the stem (\u003cstrong\u003eb\u003c/strong\u003e), stem loop (\u003cstrong\u003ec\u003c/strong\u003e), and U-rich loop (\u003cstrong\u003ed\u003c/strong\u003e). Hydrogen bonds are shown as dashed lines.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee–g\u003c/strong\u003e) Recognition of the end of the guide–target heteroduplex (\u003cstrong\u003ee\u003c/strong\u003e), the PAM duplex (\u003cstrong\u003ef\u003c/strong\u003e), and the substrate NTS within the RuvC active site (\u003cstrong\u003eg\u003c/strong\u003e). Hydrogen bonds and Mg\u003csup\u003e2+\u003c/sup\u003e coordinations are shown as dashed lines.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eh\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e DNA cleavage activities of WT Casλ2 and Casλ2 mutants. Data are mean ± s.d. (n = 3).\u003c/p\u003e","description":"","filename":"CasLambda2NatCommunMainFig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/1802b00e380ab5a5c823c5fc.jpg"},{"id":78825956,"identity":"36f29d8e-24c7-46c0-878a-0bef82616758","added_by":"auto","created_at":"2025-03-19 12:32:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":844835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structures of Casλ2 in distinct functional states.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea, c, and e\u003c/strong\u003e) Cryo-EM density maps (top) and structural models (bottom) of the Casλ2–crRNA–target DNA complexes in the catalytically incompetent (\u003cstrong\u003ea\u003c/strong\u003e), intermediate (\u003cstrong\u003ec\u003c/strong\u003e), and NTS-cleaving (\u003cstrong\u003ee\u003c/strong\u003e) states. The disordered region is indicated by a dashed circle.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb, d, and f\u003c/strong\u003e) Close-up views around the RuvC active site in the catalytically incompetent (\u003cstrong\u003eb\u003c/strong\u003e), intermediate (\u003cstrong\u003ed\u003c/strong\u003e), and NTS-cleaving (\u003cstrong\u003ef\u003c/strong\u003e) states. The disordered region is indicated by a dashed circle.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg\u003c/strong\u003e) Cryo-EM density map (left) and structural model (right) of the Casλ2–crRNA–target DNA complexes in the TS-cleaving state.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eh\u003c/strong\u003e) Recognition of the extended TS within the RuvC active site.\u003c/p\u003e","description":"","filename":"CasLambda2NatCommunMainFig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/8160a273550c140929e0da7d.jpg"},{"id":78825560,"identity":"90429b0d-3e93-4eb4-8552-157730e20e3d","added_by":"auto","created_at":"2025-03-19 12:24:00","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":863496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePre-crRNA processing by Casλ2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic of the pre-crRNAs used for the pre-crRNA processing experiments. Top, spacer-repeat-spacer (srs-type) pre-crRNA; Bottom, repeat-spacer-repeat (rsr-type) pre-crRNA.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e pre-crRNA processing activities of WT Casλ2 and dCasλ2 (D324A). While the srs-type pre-crRNA was not cleaved by Casλ2 proteins, the rsr-type pre-crRNA was efficiently cleaved by WT Casλ2, indicating that Casλ2 processes its pre-crRNA on the 3′ side by using the RuvC active site. The cleavage products were analyzed by 10% denaturing urea-PAGE.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Schematic of the pre-crRNAs used for determining the pre-crRNA processing pattern.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eIn vitro\u003c/em\u003e pre-crRNA processing activities of WT Casλ2 for pre-crRNAs with different spacer lengths (17 and 24 nt) and those with a 20-nt polyA sequence at the 3′ end. The cleavage products were analyzed by 10% denaturing urea-PAGE.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Comparison of the lengths of pre-crRNA cleavage products (resulting in repeat-spacer) with those of synthesized crRNAs with different spacer lengths.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Cryo-EM density map (left) and structural model (right) of the dCasλ2–pre-crRNA binary complex.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg\u003c/strong\u003e) Cryo-EM density map (left) and recognition of the pre-ordered spacer by Casλ2 (right). In the cryo-EM density map, crRNA is colored red.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eh\u003c/strong\u003e) Proposed model of Casλ2 pre-crRNA processing by the ruler mechanism.\u003c/p\u003e","description":"","filename":"CasLambda2NatCommunMainFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/6e96fee2ce66a1bfc2736a14.jpg"},{"id":78825555,"identity":"768afd60-2730-436f-a947-b65f1cc1fa42","added_by":"auto","created_at":"2025-03-19 12:24:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":699167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStepwise mechanisms of action of Casλ2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic showing conformational changes in Casλ2 during DNA binding and cleavage. In the binary complex, Casλ2 adopts a closed conformation, accommodating a relatively long 12-nt spacer within the central groove to protect it from being cleaved. The remaining downstream spacer is bent and spontaneously directed toward the RuvC active site, resulting in the cleavage of pre-crRNA at a position 16–18 nt downstream from the crRNA scaffold. Upon target DNA binding, Casλ2 adopts an open conformation to accommodate the guide–target heteroduplex, with the REC2 domain dissociating from the TNB domain, and part of the RuvC domain and the entire TNB domain becoming disordered (incompetent state). Subsequently, the RuvC and TNB domains become ordered, accompanying the formation of the RuvC active site, although the substrate DNA is not yet bound (intermediate state). A subsequent closing movement of the REC2 domain toward the TNB domain, along with a local conformational rearrangement of the TNB domain, allows the NTS to enter the RuvC active site (NTS-cleaving state). Cleavage of the NTS is followed by the TS being directed along the positively charged surface of the REC3 domain to the RuvC active site (TS-cleaving state). The catalytically competent RuvC active site is indicated with a yellow star.\u003c/p\u003e","description":"","filename":"CasLambda2NatCommunMainFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/f7b01a2532f9efa6b2eec76a.jpg"},{"id":78825954,"identity":"e17cf7a6-e788-40bc-a7c7-14334524b43e","added_by":"auto","created_at":"2025-03-19 12:32:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":789552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural comparison of Casλ2 with other Cas12 effectors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStructural comparisons of Casλ2 with Casλ1 (PDB: 8DC2), CasΦ (PDB: 7LYT), and Cas12a (PDB: 6I1K). Structural models are fully colored to represent their domain configurations (left) or selectively colored to highlight regions involved in recognizing the PAM-distal region of the guide–target heteroduplex (center).\u003c/p\u003e","description":"","filename":"CasLambda2NatCommunMainFig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/77e18bddd1005ee8dc693eb3.jpg"},{"id":84045277,"identity":"9035fce0-f337-440f-9c34-a26b496fe27f","added_by":"auto","created_at":"2025-06-06 07:09:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6708203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/a3e7723c-dc9e-4afd-ac13-4570c5a93b5a.pdf"},{"id":78825550,"identity":"1b134e97-7881-4888-8dd7-3d1321815555","added_by":"auto","created_at":"2025-03-19 12:23:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":202898,"visible":true,"origin":"","legend":"Supplementary Tables S1-S2","description":"","filename":"Supplementarytables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/142bda154b4bce43453cb467.pdf"},{"id":78825557,"identity":"56e0761c-495a-43e8-a864-4eab3f142475","added_by":"auto","created_at":"2025-03-19 12:24:00","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":6674959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure Legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 1 | Single-particle cryo-EM analysis of the Casλ2–crRNA–target DNA ternary complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Size-exclusion chromatography profile of the Casλ2–crRNA–target DNA complex. The peak fraction indicated as the complex was used for the following cryo-EM analyses.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Representative cryo-EM micrograph, recorded on a 300-kV Titan Krios 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. The data processing done in cryoSPARC is shown in blue, while the processing done in RELION is shown in black.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee–g\u003c/strong\u003e) Cryo-EM density maps of the Casλ2–crRNA–target DNA complexes in the catalytically incompetent (\u003cstrong\u003ee\u003c/strong\u003e), intermediate (\u003cstrong\u003ef\u003c/strong\u003e), and NTS-cleaving (\u003cstrong\u003eg\u003c/strong\u003e) states, according to the local resolution.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eh–j\u003c/strong\u003e) Fourier shell correlation curves for the 3D reconstructions of the Casλ2–crRNA–target DNA complexes in the catalytically incompetent (\u003cstrong\u003eh\u003c/strong\u003e), intermediate (\u003cstrong\u003ei\u003c/strong\u003e), and NTS-cleaving (\u003cstrong\u003ej\u003c/strong\u003e) states.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 2 | RuvC domain and crRNA structures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) The RuvC domain structure of Casλ2 and that of UnCas12f (PDB: 7C7L). The RuvC domains comprise the RNaseH fold. The conserved β strands and α helices are numbered in blue and red, respectively, while the additional helices observed in Casλ2 are numbered in green. The conserved RuvC active site is indicated as red spheres.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Schematic of the RuvC domain of Casλ2.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Structures of crRNAs of Casλ2 (left), Cas12m2 (center) (PDB: 8HHL), and Cas12a (right) (PDB: 6I1K). While the crRNAs of most of the Cas12 enzymes, including Cas12m2 and Cas12a, comprise the conserved pseudoknot structure, the crRNA of Casλ2 comprises the stem and stem-loop, but lacks the pseudoknot structure. PK, pseudoknot.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 3 | Schematic of nucleic acid recognition.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResidues that interact with nucleic acids through their main chains are shown in parentheses. The disordered regions are indicated by dashed gray lines.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 4 | Multiple sequence alignment of Casλ orthologues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultiple sequence alignment of Casλ1, Casλ2, and DAE37039 (a hypothetical protein from Bacteriophage sp. that demonstrated sequence similarity to Casλ2 in a BLAST analysis). The secondary structure of Casλ2 is indicated above the sequences. The additional helices observed in the RuvC domain of Casλ2 are numbered in green, as in Figure S2A. The key residues of Casλ2 are marked with triangles below the sequences. The figure was prepared using Clustal Omega (https://www.ebi.ac.uk/jdispatcher/msa/clustalo) and ESPript3 (https://espript.ibcp.fr/ESPript/ESPript/).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 5 | Map-model superimposition and conformational changes of Casλ2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Overall cryo-EM density map (left) and map-model superimposition of the substrate NTS bound within the RuvC active site (right).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Close-up views of the RuvC active site of Casλ2 in the NTS-cleaving state. Cryo-EM density maps are shown as translucent gray surfaces. The bound Mg\u003csup\u003e2+\u003c/sup\u003e ions are shown as green spheres.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec and d\u003c/strong\u003e) Structures (top) and close-up views around the RuvC domain (bottom) of the Casλ2–crRNA–target DNA complexes in the intermediate (\u003cstrong\u003eC\u003c/strong\u003e) and NTS-cleaving (\u003cstrong\u003eD\u003c/strong\u003e) states. Hydrogen bonds are shown as dashed lines.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Superimposition of the intermediate (orange) and NTS-cleaving (colored as in \u003cstrong\u003eD\u003c/strong\u003e) states. The top and bottom panels display the overall structure and a close-up view around the RuvC active site, respectively. The inward movement of the REC2 domain toward the TNB domain is accompanied by the inward movement of a helix in the TNB domain toward the REC2 domain. The substrate NTS in the NTS-cleaving state clashes with the lid loop in the intermediate state.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 6 | Single-particle cryo-EM analysis of the Casλ2–crRNA–target DNA ternary complex in the TS-cleaving state.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Schematic of the target DNA construct used in the complex reconstruction. Phosphorothioate modified regions in the target DNA strand are shown in green. In this panel, please change phosphorthioate to phosphorothioate.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Size-exclusion chromatography profile of the Casλ2–crRNA–target DNA complex in the TS-cleaving state. The peak fraction indicated as the complex was used for the following cryo-EM analyses.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ec\u003c/strong\u003e) Representative cryo-EM micrograph, recorded on a 300-kV Titan Krios microscope with a K3 camera.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ed\u003c/strong\u003e) Single-particle cryo-EM image processing workflow. The data processing done in cryoSPARC is shown in blue, while the processing done in RELION is shown in black.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Fourier shell correlation curve for the 3D reconstruction.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Cryo-EM density map according to the local resolution.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended Data Fig. 7 | Single-particle cryo-EM analysis of the dCasλ2–pre-crRNA binary complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Size-exclusion chromatography profile of the dCasλ2–pre-crRNA complex. The peak fraction indicated as the complex was used for the following cryo-EM analyses.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Representative cryo-EM micrograph, recorded on a 300-kV Titan Krios 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. The data processing done in cryoSPARC is shown in blue, while the processing done in RELION is shown in black.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ee\u003c/strong\u003e) Fourier shell correlation curve for the 3D reconstruction.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ef\u003c/strong\u003e) Cryo-EM density map according to the local resolution.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eg and h\u003c/strong\u003e) Structures (top) and close-up views (bottom) of the Casλ2–crRNA–target DNA complex in the NTS-cleaving state (\u003cstrong\u003eg\u003c/strong\u003e) and the dCasλ2–pre-crRNA binary complex (\u003cstrong\u003eh\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ei\u003c/strong\u003e) Superimposition of the NTS-cleaving state (orange) and the binary complex (colored as in \u003cstrong\u003eh\u003c/strong\u003e). The top and bottom panels display the overall structure and a close-up view, respectively.\u003c/p\u003e","description":"","filename":"CasLambda2NatCommunExtendedFig.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/9ba507b024aa415ae7b954b4.pdf"},{"id":78825564,"identity":"b9fb37e4-f711-4d13-8983-0fd81b13a59b","added_by":"auto","created_at":"2025-03-19 12:24:05","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":74366463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Movie Legend\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Movie S1 | Conformational rearrangements of Casλ2 during its nuclease activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStepwise domain rearrangements of Casλ2.\u003c/p\u003e","description":"","filename":"SupplementaryMovieS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5481685/v1/30892f2f1cced631c594f49c.mp4"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nG.M., A.J.G., A.O., L.E.A., G.R.H., T.D., W.X.Y., D.R.C., D.A.S., and Z.M. are current or former employees and shareholders of Arbor Biotechnologies. Arbor Biotechnologies has filed patents related to this work. O.N. is a co-founder, board member, and scientific advisor of Curreio. The remaining authors declare no competing interests.","formattedTitle":"Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems provide adaptive immunity against mobile genetic elements (MGEs) in prokaryotes and are divided into two classes (classes 1 and 2) and six types (types I\u0026ndash;VI)\u003csup\u003e1,2\u003c/sup\u003e. Class 2 systems include types II, V, and VI, in which Cas9, Cas12, and Cas13, respectively, function as single, multidomain effectors to interfere with MGEs. Cas9 from \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e (SpCas9) associates with dual RNA guides (CRISPR RNA [crRNA] and trans-activating crRNA [tracrRNA]) and cleaves double-stranded DNA (dsDNA) targets at sequences complementary to a 20-nt guide segment in the RNA guide and flanked by NGG (where N is any nucleotide) protospacer adjacent motifs (PAMs), using its RuvC and HNH nuclease domains\u003csup\u003e3,4\u003c/sup\u003e. By contrast, Cas12a from \u003cem\u003eAcidaminococcus sp.\u003c/em\u003e (AsCas12a) associates with a crRNA and cleaves dsDNA targets at sequences complementary to a 20-nt guide segment with TTTV (where V is A, G, or C) PAMs, using a single RuvC nuclease domain\u003csup\u003e5\u003c/sup\u003e. Since SpCas9 and AsCas12a exhibit robust DNA cleavage activities in eukaryotic cells, they are widely utilized as powerful genome engineering tools\u003csup\u003e5,6\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough CRISPR-Cas systems are typically encoded in the genomes of bacteria and archaea, recent studies have described the widespread presence of diverse types of CRISPR-Cas systems encoded in bacteriophage genomes\u003csup\u003e7\u0026ndash;11\u003c/sup\u003e. Among them, single multidomain CRISPR-Casλ effector proteins have been identified as compact RNA-guided DNA endonucleases\u003csup\u003e11\u003c/sup\u003e. The Mahaphage-encoded Casλ1 protein, consisting of 747 amino acid residues, is about half the size of SpCas9 and AsCas12a. Casλ1 binds to a crRNA and cleaves dsDNA targets with TTR (where R is A or G) PAMs, using its single RuvC nuclease domain\u003csup\u003e11\u003c/sup\u003e. Moreover, Casλ1 processes its cognate precursor crRNA (pre-crRNA) to the mature crRNA by using its RuvC domain, as observed in Cas12c and Cas12j (also known as CasΦ)\u003csup\u003e10\u0026ndash;13\u003c/sup\u003e. Despite bearing little sequence similarity to known Cas12 proteins, these biochemical features of Casλ1 suggested its classification as a phage-encoded type V CRISPR-Cas effector\u003csup\u003e11\u003c/sup\u003e. However, the diversity and conservation of the biochemical properties among Casλ family enzymes are unclear, and the detailed molecular basis of DNA cleavage and guide RNA processing by Casλ remain enigmatic.\u003c/p\u003e \u003cp\u003eHere, using metagenomic and phylogenetic analyses, we identified CRISPR-Casλ2 as a member of the Casλ family of enzymes. Biochemical analysis revealed the robust dsDNA cleavage activity of Casλ2, with an optimal 16\u0026ndash;18-nt guide crRNA under physiological conditions, creating asymmetric staggered DNA double-stranded breaks distinct from the cleavage pattern observed with Casλ1. In addition, we showed that Casλ2 processes its pre-crRNA in a unique manner, in which Casλ2 functions as a \u0026ldquo;molecular ruler\u0026rdquo; to define the spacer lengths of the mature crRNA. We then determined the cryo-electron microscopy (cryo-EM) structures of Casλ2 in complex with the crRNA and dsDNA target in four distinct functional states, and with the pre-crRNA in one state, illustrating the stepwise domain rearrangements occurring during nuclease activation. Overall, this study advances our understanding of the molecular mechanisms of diverse type V CRISPR-Cas effectors and establishes a foundation for future engineering of Casλ enzymes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of the CRISPR-Casλ2 system\u003c/h2\u003e \u003cp\u003eA discovery campaign was previously initiated to identify diverse CRISPR-Cas systems, resulting in a database of candidate effectors\u003csup\u003e14,15\u003c/sup\u003e. From this database, we identified a putative type V Cas variant with an open reading frame (ORF) size of 741 amino acids and a unique RuvC domain within its C-terminal region. Metagenomic tracing confirmed that this effector was first isolated in a microbiome sample from \u003cem\u003eBos taurus\u003c/em\u003e, raised in Alberta, CA\u003csup\u003e16\u003c/sup\u003e. Phylogenetic analysis revealed that this newly identified variant clustered with a previously reported CRISPR-Casλ effector encoded by bacteriophage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Based on this similarity, we designated this variant as CRISPR-Casλ2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo functionally characterize the CRISPR-Casλ2 system, we employed an \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) negative selection screen as described previously\u003csup\u003e14,15\u003c/sup\u003e. Briefly, we prepared a plasmid carrying the Casλ2 gene, a CRISPR array library targeting pACYC184 and essential \u003cem\u003eE. coli\u003c/em\u003e genes, and concatenated \u003cem\u003ecas\u003c/em\u003e gene-flanking noncoding sequences for the unbiased detection of a tracrRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The plasmid was transformed into \u003cem\u003eE. coli\u003c/em\u003e cells which were then grown under antibiotic selection. In screening strategy, when effector RNA-guided interference activity is present, bacterial viability is reduced. This reduction is detected by quantification of array depletion in the harvested cells, which revealed that Casλ2 demonstrated robust interference activity against dsDNA targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Similar activity was observed in both the presence and absence of the noncoding flanking sequences from the effector-encoding plasmid, indicating that Casλ2 efficiently targets dsDNA and functions with the cognate crRNA without the requirement of a tracrRNA.\u003c/p\u003e \u003cp\u003eTo identify the PAM specificity of Casλ2, we assessed the target-flanking sequences of the strongly depleted arrays in the \u003cem\u003eE. coli\u003c/em\u003e screens. Deep sequencing analysis revealed that the dsDNA interference by Casλ2 depends on 5\u0026prime;-TTR-3\u0026prime; (where R represents A or G) PAMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Subsequently, we investigated the crRNA-guided DNA cleavage activity of Casλ2 through \u003cem\u003ein vitro\u003c/em\u003e DNA cleavage assays, using purified Casλ2, a crRNA with a 20-nt guide segment, and linearized plasmid DNA containing a target sequence and 5\u0026prime;-TTN-3\u0026prime; PAMs. Casλ2 did not cleave target DNAs with 5\u0026prime;-TTY-3\u0026prime; (where Y represents T or C) PAMs, but efficiently cleaved target DNAs with 5\u0026prime;-TTR-3\u0026prime; PAMs, exhibiting slightly higher activity with the 5\u0026prime;-TTA-3\u0026prime; PAM compared to the 5\u0026prime;-TTG-3\u0026prime; PAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Collectively, these results indicate that CRISPR-Casλ2 is a variant of the phage-encoded CRISPR-Casλ family and exhibits crRNA-guided dsDNA cleavage activity with a 5\u0026prime;-TTR-3\u0026prime; PAM.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiochemical characterization of Casλ2-mediated dsDNA cleavage\u003c/h3\u003e\n\u003cp\u003eTo comprehensively assess the biochemical properties of Casλ2, we examined the \u003cem\u003ein vitro\u003c/em\u003e DNA cleavage activity of purified Casλ2 using a linearized plasmid DNA target bearing a 5\u0026prime;-TTA-3\u0026prime; PAM under various biochemical conditions. Casλ2 efficiently cleaved the target DNA over a temperature range of 37 to 55\u0026deg;C, with a preference for NaCl concentrations between 25 and 100 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee and f). Casλ2 required spacer lengths of at least 16 nucleotides for effective dsDNA cleavage, with 16\u0026ndash;18-nt guide crRNAs being optimal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eCasλ1 was previously reported to cleave the target DNA strand (TS) and non-target DNA strand (NTS) at 11\u0026ndash;13 nt and 26\u0026ndash;29 nt downstream of the PAM, respectively, generating cleavage products with pronounced staggered 5\u0026prime;-overhangs of 11\u0026ndash;16 nt\u003csup\u003e11\u003c/sup\u003e. In contrast, Sanger sequencing of the target DNA cleavage products revealed that Casλ2 cleaved the TS at 23 nt downstream of the PAM and NTS at 19 or 20-nt downstream of the PAM, generating 3 or 4-nt 5\u0026prime;-overhangs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh).\u003c/p\u003e\n\u003ch3\u003eCryo-EM structure of the Casλ2–crRNA–target DNA ternary complex\u003c/h3\u003e\n\u003cp\u003eTo understand the RNA-guided DNA cleavage mechanism of Casλ2, we determined the cryo-EM structure of Casλ2 in complex with a 56-nt crRNA and its 40-nt dsDNA target bearing the 5\u0026prime;-TTA-3\u0026prime; PAM, at an overall resolution of 2.9 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and Supplementary Tables S1 and S2). To prevent target DNA cleavage during the reconstruction, we used dsDNA with phosphorothioate modifications within the DNA backbone around the cleavage site. Casλ2 adopts a bilobed architecture consisting of recognition (REC) and nuclease (NUC) lobes, as observed in the structure of Casλ1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;c). The REC lobe consists of the WED, REC1, and REC2 domains, while the NUC lobe has the RuvC, REC3, and target nucleic acid-binding (TNB) domains. The WED domain adopts a typical oligonucleotide/oligosaccharide-binding (OB) fold, consisting of seven β strands flanked by an α helix. The REC1 and REC2 domains comprise six α helices and four α helices, respectively, with each forming a characteristic globular shape.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe RuvC domain has an RNaseH fold, consisting of a conserved five-stranded mixed β sheet flanked by four α helices, with Asp324, Glu526, and Asp680 forming an active center similar to those of other Cas12 enzymes (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b)\u003csup\u003e13,17\u0026ndash;22\u003c/sup\u003e. An α helix (referred to as the lid helix) is inserted between strand β4 and helix α3, corresponding to the region known as the lid motif in other Cas12 enzymes. Additionally, the RuvC domain of Casλ2 has unique insertion helices between the strands β2 and β3, and between the strand β3 and helix α1, expanding the size of the RuvC domain as compared with other Cas12 enzymes (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b). The REC3 domain, which is not resolved in the previously reported Casλ1 structure, contains a two-stranded antiparallel β-sheet and two α helices, and is inserted between the strand β4 and lid helix of the RuvC domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). A Dali search revealed that the REC3 domain of Casλ2 lacks structural similarity with any other known proteins, including those of Cas12 enzymes\u003csup\u003e23\u003c/sup\u003e. The TNB domain comprises a three-stranded mixed β-sheet and two α helices.\u003c/p\u003e \u003cp\u003eThe guide\u0026ndash;target heteroduplex is accommodated within the central channel between the REC and NUC lobes, whereas the PAM-containing DNA duplex (PAM duplex) is sandwiched by the WED and REC1 domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and c). The crRNA scaffold is bound within the groove formed by the WED and RuvC domains. Notably, the substrate single-stranded DNA (ssDNA) of the 3\u0026prime; NTS is bound within the RuvC active site, which is not observed in the Casλ1 structure, indicating that the current structure represents a catalytically active state, as described below.\u003c/p\u003e\n\u003ch3\u003ecrRNA architecture and recognition\u003c/h3\u003e\n\u003cp\u003eThe crRNA consists of the 20-nt guide segment (G1 to C20) and the 36-nt repeat-derived scaffold (C(\u0026minus;\u0026thinsp;36) to C(\u0026minus;\u0026thinsp;1)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and e). Notably, the crRNA scaffold comprises the stem and stem-loop, and lacks the pseudoknot structure conserved within most Cas12 enzymes (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec)\u003csup\u003e13,17\u0026ndash;20,24,25\u003c/sup\u003e. The stem contains a 5-bp duplex (G(\u0026minus;\u0026thinsp;33):C(\u0026minus;\u0026thinsp;1) to U(\u0026minus;\u0026thinsp;29):A(\u0026minus;\u0026thinsp;5)), while the stem-loop consists of a 6-bp distorted duplex (A(\u0026minus;\u0026thinsp;28):U(\u0026minus;\u0026thinsp;8) to U(\u0026minus;\u0026thinsp;20):A(\u0026minus;\u0026thinsp;14)) with a wobble base pair (U(\u0026minus;\u0026thinsp;23):G(\u0026minus;\u0026thinsp;12)) and two flipped-out bases (U(\u0026minus;\u0026thinsp;27) and U(\u0026minus;\u0026thinsp;22)), connected by a characteristic U-rich loop (U(\u0026minus;\u0026thinsp;19) to U(\u0026minus;\u0026thinsp;15)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The stem and stem-loop are connected with an approximate 80\u0026deg; bend, mediated by a two-adenine junction (A(\u0026minus;\u0026thinsp;7) and A(\u0026minus;\u0026thinsp;6)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eThe crRNA scaffold is recognized by Casλ2 through both base-specific and non-specific interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The stem is accommodated within the groove formed by the WED and RuvC domains, and primarily recognized by these domains through interactions with its sugar-phosphate backbone. In particular, the first G(\u0026minus;\u0026thinsp;33):C(\u0026minus;\u0026thinsp;1) base pair of the stem is recognized by Arg271 and Asn625 through hydrogen bonds, while the phosphate backbones of U(\u0026minus;\u0026thinsp;32) and G1, adjacent to the G(\u0026minus;\u0026thinsp;33):C(\u0026minus;\u0026thinsp;1) base pair, hydrogen bond with Lys8 and Tyr709, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The stem-loop is exposed to the solvent and has minimal interactions with the RuvC domain, with helix α5 inserted within the RuvC domain playing central roles in the stem-loop recognition. Specifically, Gln412, Lys413, and Lys416 form hydrogen bonds with the backbone phosphate group of U(\u0026minus;\u0026thinsp;27) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Tyr409 stacks with U(\u0026minus;\u0026thinsp;27) and hydrogen bonds with the phosphate group of A(\u0026minus;\u0026thinsp;14) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The U-rich loop is recognized by the RuvC domain through base-specific interactions. Specifically, U(\u0026minus;\u0026thinsp;17) is stabilized by coordination with the magnesium ion and forms base-specific hydrogen bonds with Lys436 and Asn495, suggesting that the U-rich loop plays an important role in the sequence-specific recognition of the crRNA by Casλ2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Collectively, these structural observations reveal how Casλ2 assembles with its cognate crRNA to form a ribonucleoprotein complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eTarget DNA architecture and recognition\u003c/h3\u003e\n\u003cp\u003eThe target DNA forms a 17-bp guide\u0026ndash;target heteroduplex and 10-bp PAM duplex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Nucleotides dG4* to dG18* and dT25* to dT30* in the NTS and dA(\u0026minus;\u0026thinsp;13) to dC(\u0026minus;\u0026thinsp;1) in the TS are not resolved in the density map, probably due to their flexibility. The guide\u0026ndash;target heteroduplex is accommodated within the positively charged central groove formed by the REC1, REC2, REC3, and RuvC domains, and is recognized by these domains mainly through sugar-phosphate backbone interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Lys2, which is not conserved in Casλ1, forms a hydrogen bond with the backbone phosphate group between dC17 and dT18 and a hydrophobic interaction with the first G1:dC17 base pair in the heteroduplex, suggesting that Casλ2 facilitates target DNA unwinding in a distinct manner from Casλ1 (Extended Data Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, Tyr548 in the REC3 domain stacks with the U17:dA1 base pair in the heteroduplex to preclude the extension of the heteroduplex beyond the protein, as observed in other Cas12 enzymes, explaining the 16\u0026ndash;18-nt preference for Casλ2-mediated DNA cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the present structure, the PAM duplex with the 5\u0026prime;-TTA-3\u0026prime; PAM is bound between the WED and REC1 domains and recognized in a base-specific manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Specifically, the 5-methyl groups of dT(\u0026minus;\u0026thinsp;3*) and dT(\u0026minus;\u0026thinsp;2*) in the TTR PAM form hydrophobic interactions with Thr113 and Phe118, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The dA20 nucleobase, which base pairs with dT(\u0026minus;\u0026thinsp;3*), forms base-specific hydrogen bonds with Gln239 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The N7 atom of dA(\u0026minus;\u0026thinsp;1*) hydrogen bonds with Asn89, explaining the requirement for the third R nucleotide in the TTR PAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). In addition, the 5-methyl group of the dT18 nucleobase, which base pairs with dA(\u0026minus;\u0026thinsp;1*), forms hydrophobic interactions with Phe118 and Ile237, providing an explanation for the slight preference of Casλ2 for the TTA PAM rather than the TTG PAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Indeed, alanine substitutions of Asn89, Thr113, Phe118, Ile237, and Gln239 reduced or abolished the Casλ2-mediated DNA cleavage activity \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). Taken together, these structural and biochemical observations reveal the mechanism of PAM recognition by Casλ2.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSubstrate DNA recognition within the RuvC active site\u003c/h2\u003e \u003cp\u003eIn the Casλ2 structure, we observed a 6-nt ssDNA density bound within the groove between the RuvC and TNB domains, which is not observed in the Casλ1 structure (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Nucleotides dG19* to dA24* in the NTS are well fitted in the density map and enter the RuvC catalytic site, indicating that the present structure likely represents the NTS-cleaving state (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The substrate NTS is recognized by Casλ2 in a sequence-independent manner. Notably, Tyr178 in the REC2 domain forms a hydrogen bond with the backbone phosphate of dG19*, while Pro721 in the TNB domain stacks with the base of dG19*, thereby anchoring dG19* at the entrance of the RuvC active site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). In addition, Phe533 in the RuvC domain inserts between dC20* and dA21* and forms stacking interactions with their nucleobases, kinking the substrate DNA around a scissile phosphate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Tyr178 and Phe533 are well conserved in Casλ enzymes and their alanine substitutions reduced or abolished the \u003cem\u003ein vitro\u003c/em\u003e DNA cleavage activities of Casλ2, confirming the importance of these residues for substrate DNA recognition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe phosphate group of dA21* is bound to the RuvC active site via two magnesium ions (Mg\u003csub\u003eA\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csub\u003eB\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e, respectively), which are likely coordinated by Asp324, Glu526, and Asp680 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Additionally, Asn326 in the β1 strand of the RuvC domain participates in the coordination of Mg\u003csub\u003eA\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The alanine substitution of Asn326 almost completely eliminated the DNA cleavage activity, suggesting the important role of Asn326 for magnesium ion coordination besides the typical DED catalytic triad (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). These structural and biochemical findings indicate that Casλ2 cleaves the target DNA in a manner dependent on two magnesium ions, as observed in other Cas12 enzymes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConcomitant conformational changes of Casλ2 for target DNA cleavage\u003c/h3\u003e\n\u003cp\u003eIn addition to the NTS-cleaving state (State III), we found two distinct classes (States I and II) during the cryo-EM data analysis, and determined the two additional structures at overall resolutions of 2.9 (State I) and 3.1 (State II) \u0026Aring; (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although Casλ2 recognizes the target DNA and crRNA similarly in all three states, we observed notable structural differences in their REC2, RuvC, and TNB domains.\u003c/p\u003e \u003cp\u003eIn State I, Casλ2 adopts an open conformation, where the REC2 domain is distant from the NUC lobe with low local resolution, probably due to its flexibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Furthermore, in this state, part of the RuvC domain and the entire TNB domain (residues 633 to 741) are not resolved in the density map (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and b). These structural observations suggest that this structure likely represents a \u0026ldquo;catalytically incompetent state\u0026rdquo;, in which the RuvC and TNB domains are flexible and the RuvC active site has not yet been formed.\u003c/p\u003e \u003cp\u003eIn contrast, in State II, the RuvC and TNB domains are fully resolved in the density map, while the REC2 domain remains distant from the NUC lobe (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In this state, although the RuvC active site is located at a position similar to that in the NTS-cleaving state, the substrate DNA strand is not bound within the RuvC domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Thus, this structure represents an \u0026ldquo;intermediate state\u0026rdquo;, in which the RuvC and TNB domains have become ordered but not yet adopted the proper local conformation for accommodating the substrate DNA strand within the groove between the RuvC and TNB domains.\u003c/p\u003e \u003cp\u003eA structural comparison between the intermediate and NTS-cleaving states reveals the local conformational rearrangements of Casλ2 for accommodating the substrate DNA within the RuvC active site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u0026ndash;f and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u0026ndash;e). In the intermediate state, residues 527 to 540 (referred to as the lid loop here) are distant from the guide\u0026ndash;target heteroduplex and occlude the RuvC active site (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In the NTS-cleaving state, in contrast, Lys535 in the lid loop forms a hydrogen bond with the backbone phosphate of G11 in the heteroduplex, thereby facilitating the structural transition of the lid loop to open the RuvC active site (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Additionally, we observed a concomitant conformational rearrangement between the REC2 and TNB domains. During the transition from the intermediate state to the NTS-cleaving state, the REC2 domain undergoes a significant movement of approximately 30 \u0026Aring; toward the TNB domain (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). This conformational change cooperatively induces the inward movement of a helix in the TNB domain toward the REC2 domain, providing interaction interfaces between the REC2 and TNB domains (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Specifically, Tyr178 in the REC2 domain and Pro721 in the TNB domain form a stacking interaction, while Leu725 and His726 in the TNB domain form hydrophobic and hydrogen-bonding interactions with Tyr178 and Glu174 in the REC2 domain, respectively, thereby stabilizing the closed conformation of Casλ2 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and e). This closed conformation enables Tyr178 and Pro721 to interact with the substrate DNA strand bound within the RuvC active site, as described above. Taken together, these structural observations demonstrate the concerted conformational changes of the REC2, RuvC, and TNB domains for target DNA cleavage by Casλ2.\u003c/p\u003e\n\u003ch3\u003eTarget strand cleavage by Casλ2\u003c/h3\u003e\n\u003cp\u003ePrevious studies revealed that Cas12 enzymes cleave their dsDNA targets by using the single RuvC nuclease domain in a sequential manner, in which NTS cleavage is followed by TS cleavage\u003csup\u003e26\u0026ndash;28\u003c/sup\u003e. To elucidate the TS cleavage mechanism of Casλ2, we used a 14-nt NTS and a 40-nt TS during complex reconstruction to mimic the post-NTS-cleavage state, and determined the ternary complex structure at an overall resolution of 2.8 \u0026Aring; (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Although the overall structure is similar to the NTS-cleaving state, we observed the extended TS density entering the RuvC active site, suggesting that this structure represents the TS-cleaving state (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). The extended TS is kinked at the end of the guide\u0026ndash;target heteroduplex and folded back along the REC3 domain to access the RuvC domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe single-stranded segment of the TS beyond the guide\u0026ndash;target heteroduplex is recognized by Casλ2 in an almost identical manner to that observed in the NTS-cleaving state. Tyr178 and Pro721 form hydrogen-bonding and stacking interactions with dT(\u0026minus;\u0026thinsp;7), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Phe533 forms stacking interactions with dG(\u0026minus;\u0026thinsp;6) and dG(\u0026minus;\u0026thinsp;5), kinking the TS around the scissile phosphate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). The phosphate group of dG(\u0026minus;\u0026thinsp;5), located 23 nt downstream from the PAM, is bound to the RuvC active site with two magnesium ions, coordinated by Asp324, Asn326, Glu526, and Asp680, explaining the TS cleavage position 23-nt downstream from the PAM observed in our biochemical analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Together, these structural observations indicate that Casλ2 cleaves both the NTS and TS in an identical manner within the RuvC active site, with the REC3 domain providing a pathway for the TS toward the RuvC active site.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGuide RNA processing by the Casλ2\u003c/h2\u003e \u003cp\u003eRecent studies have shown that Casλ1 processes its pre-crRNA at the spacer region by using the RuvC active site\u003csup\u003e11\u003c/sup\u003e. To determine whether Casλ2 is also capable of processing its pre-crRNA into the mature crRNA, we performed an \u003cem\u003ein vitro\u003c/em\u003e pre-crRNA processing assay using two types of pre-crRNAs, composed of a 5\u0026prime;-spacer\u0026ndash;repeat\u0026ndash;spacer-3\u0026prime; (srs) or 5\u0026prime;-repeat\u0026ndash;spacer\u0026ndash;repeat-3\u0026prime; (rsr) sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The rsr-type pre-crRNA was successfully processed by Casλ2, while the srs-type pre-crRNA was not (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). We also found that the D324A mutant (dCasλ2) lost the pre-crRNA processing activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Together, these results indicate that Casλ2 cleaves the 3\u0026prime; side of the pre-crRNA by using the RuvC active site, as observed in Casλ1.\u003c/p\u003e \u003cp\u003eIn addition to the Casλ enzymes, Cas12c and Cas12j also process their pre-crRNAs at their RuvC active sites through distinct mechanisms\u003csup\u003e10,13\u003c/sup\u003e. Cas12c recognizes both the upstream and downstream repeat sequences of the pre-crRNA and cleaves immediately upstream of the downstream repeat sequence, whereas Cas12j cleaves within the upstream spacer region of the pre-crRNA. To clarify the pre-crRNA processing mechanism by Casλ2 in detail, we prepared the rsr-type pre-crRNA with different lengths of spacers (17 nt and 24 nt, respectively) and examined the processing patterns of Casλ2 against these pre-crRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Casλ2 successfully cleaved both pre-crRNAs. Notably, while the lengths of the cleaved repeat\u0026ndash;spacer segments were not changed, the lengths of the cleaved downstream repeats changed depending on the spacer length of the pre-crRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). We also conducted \u003cem\u003ein vitro\u003c/em\u003e processing assays using a pre-crRNA-polyA substrate, in which the downstream repeat was replaced with a 20-nt polyA sequence, and observed similar processing patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). These results indicated that, unlike Cas12c and Cas12j, Casλ2 recognizes the upstream repeat sequence and cleaves the spacer region by a unique ruler mechanism, in which Casλ2 functions as a \u0026ldquo;molecular ruler\u0026rdquo; to define the spacer lengths of the mature crRNA.\u003c/p\u003e \u003cp\u003eNext, to examine the spacer length of the mature crRNA defined by Casλ2, we compared the lengths of the cleaved repeat\u0026ndash;spacer products with those of synthesized crRNAs with different spacer lengths. A denaturing urea polyacrylamide gel electrophoresis analysis revealed that the spacer length of the mature crRNA varies between 16\u0026ndash;18 nt, which is consistent with the optimal spacer length of approximately 16\u0026ndash;18 nt for target DNA cleavage by Casλ2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Collectively, our biochemical data indicate that Casλ2 processes its pre-crRNA through a ruler mechanism, in which Casλ2 recognizes the upstream repeat sequence and cleaves downstream of the recognition site at a defined length of 16\u0026ndash;18 nt.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCryo-EM structure of the Casλ2\u0026ndash;crRNA binary complex\u003c/h2\u003e \u003cp\u003eTo obtain mechanistic insights into the pre-crRNA processing by the Casλ2 ruler mechanism, we determined the cryo-EM structure of Casλ2 (the inactive D324A mutant) in complex with a pre-crRNA-polyA (76 nt) at an overall resolution of 3.0 \u0026Aring; (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, f, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u0026ndash;f, and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In the binary complex structures of most Cas12 enzymes, a 5\u0026ndash;7 nt spacer is ordered in an A-form geometry as a seed\u003csup\u003e13,18,21,29\u003c/sup\u003e. In contrast, the binary complex structure of Casλ2 revealed that the relatively long 12-nt spacer (G1 to G12) is ordered within the positively charged central groove in a stretched conformation, while nucleotides C13 to A40 are disordered probably due to their flexibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). The 12-nt spacer is extensively recognized by Casλ2 in a sequence-independent manner. Specifically, Tyr709, His230, Phe222, Tyr448, and Tyr548 form stacking interactions with G1, A5, U6, A8, and U11, respectively, thereby anchoring the 12-nt spacer in a stretched conformation rather than an A-form geometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). These structural observations suggest that Casλ2 protects a 12-nt spacer from enzymatic cleavage by accommodating it within its central groove. Subsequently, the remaining downstream spacer is bent and spontaneously directed toward the RuvC active site, resulting in pre-crRNA cleavage at a position 16\u0026ndash;18 nt downstream from the crRNA scaffold (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA structural comparison between the binary and ternary (NTS-cleaving state) complexes revealed structural rearrangements in the REC1 and REC2 domains upon target DNA binding (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg\u0026ndash;i). The ternary complex adopts an open conformation, in which the REC1 domain is distant from the RuvC domain to form a binding groove for the guide\u0026ndash;target heteroduplex. In contrast, the binary complex adopts a closed conformation, ensuring mutual interactions between the protein and 12-nt spacer. Notably, Phe222 and His230 undergo 5-\u0026Aring; and 14-\u0026Aring; shifts downward toward the RuvC domain, respectively, to form stacking interactions with the spacer (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg\u0026ndash;i). In addition, in the binary complex, the REC2 domain interacts with the TNB domain, as also observed in the NTS-cleaving state, suggesting that the binary complex is already prepared to accommodate the single-stranded RNA within the RuvC active site for pre-crRNA processing. Together, these structural observations illustrate how Casλ2 assembles with the pre-crRNA prior to the target DNA binding and functions as a molecular ruler to define the spacer length of the mature crRNA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified Casλ2 through a metagenomic analysis as a compact CRISPR-Cas effector belonging to the same branch as Casλ1, a type V CRISPR-Cas effector identified in phage genomes. Our biochemical data demonstrated that Casλ2 exhibits both crRNA-guided DNA cleavage and pre-crRNA processing activities, using a single RuvC active site. Notably, by accommodating a relatively long 12-nt spacer within the central groove, Casλ2 functions as a molecular ruler to define the spacer lengths of the mature crRNA. Furthermore, we determined the cryo-EM structures of Casλ2 in five distinct states, shedding light on the activation mechanism of Casλ enzymes.\u003c/p\u003e \u003cp\u003eA recent structural study revealed the crRNA-guided DNA binding mechanism of Casλ1\u003csup\u003e11\u003c/sup\u003e. However, the structure represents the inactive state wherein the substrate DNA is not bound within the RuvC active site, and thus the activation mechanism of Casλ family enzymes remained elusive. The cryo-EM structures of the Casλ2\u0026ndash;crRNA binary complex and the Casλ2\u0026ndash;crRNA\u0026ndash;target DNA ternary complexes in four different functional states illuminated significant conformational rearrangements during the nuclease activation of Casλ2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Supplementary Movie S1). In the binary complex, Casλ2 adopts a closed conformation, accommodating a relatively long 12-nt spacer within the central groove. Upon target DNA binding after pre-crRNA processing, Casλ2 transitions to an open conformation to accommodate the guide\u0026ndash;target heteroduplex, with the REC2 domain dissociating from the TNB domain (incompetent state). In this incompetent state, the RuvC active site is not formed due to disorder in part of the RuvC domain and the entire TNB domain. Subsequently, the RuvC and TNB domains become ordered, accompanying the formation of the RuvC active site (intermediate state). In the intermediate state, the REC2 domain remains distant from the TNB domain, and the substrate DNA is not yet bound to the RuvC active site. Then, a substantial movement of the REC2 domain toward the TNB domain, accompanied by a local conformational rearrangement of the TNB domain, enables the NTS to enter the RuvC active site (NTS-cleaving state). Cleavage of the NTS is followed by the TS being directed along the positively charged surface of the REC3 domain to the RuvC active site (TS-cleaving state). These structural observations underscore the essential domain movements and conformational rearrangements in the activation of Casλ2.\u003c/p\u003e \u003cp\u003eA structural comparison of Casλ2 with Casλ1 highlighted the mechanistic conservation and differences among the Casλ family enzymes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Although Casλ1 and Casλ2 share limited sequence similarity (37% identity), their protein structures show a high degree of structural homology, with a root mean square deviation of 0.912 \u0026Aring; across 476 pruned atom pairs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The crRNA scaffold adopts almost identical configurations in both complexes, being accommodated within the groove between WED and RuvC domains in similar manners. The PAM duplex is commonly bound between the WED and REC1 domains. In contrast to these conserved structural features, there is a notable structural difference in PAM-distal guide\u0026ndash;target heteroduplex recognition. Casλ2 recognizes a 17-bp guide\u0026ndash;target heteroduplex, with the RuvC-inserted REC3 domain playing pivotal role in distinguishing the 17th base pair. In contrast, the REC3 domain in the Casλ1 structure is disordered, accommodating a shorter 16-bp guide\u0026ndash;target heteroduplex within the central channel (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Although further biochemical characterization is needed, these structural observations suggest that Casλ2 identifies the target DNA more strictly than Casλ1 by recognizing the end of the heteroduplex through the REC3 domain.\u003c/p\u003e \u003cp\u003eStructural comparisons of Casλ2 with CasΦ enhanced our understanding of the conserved structural features among the phage-encoded type V CRISPR-Cas effectors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Despite their limited sequence similarity, both enzymes have similar sizes (CasΦ: 757 amino acids, Casλ2: 741 amino acids) and commonly adopt a bilobed architecture consisting of the REC and NUC lobes, with the guide\u0026ndash;target heteroduplex accommodated within the central channel. Both enzymes do not require tracrRNAs and rely solely on crRNAs, which adopt a simple stem-loop configuration lacking a pseudoknot structure. Recent studies suggested that, during their evolution from TnpB, Cas12 enzymes acquired the ability to recognize the PAM-distal region of the guide\u0026ndash;target heteroduplex through the insertion of the REC2 domain between their REC1 and WED domains (as seen in Cas12a), or through dimerization\u003csup\u003e25,30\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Consistent with this hypothesis, CasΦ and Casλ2 recognize longer 18- and 17-bp guide\u0026ndash;target heteroduplexes, respectively, compared to the 12-bp heteroduplex observed in the TnpB structure. Intriguingly, unlike most Cas12 enzymes, CasΦ and Casλ2 recognize the PAM-distal region of the guide\u0026ndash;target heteroduplex through the inserted region within the RuvC domain (REC3 domain in Casλ2), rather than through the REC2 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Instead, the REC2 domain of Casλ2 recognizes the substrate DNA within the RuvC active site. These findings imply that the phage-encoded type V CRISPR-Cas effectors have evolved from TnpB independently of those encoded by bacteria or archaea.\u003c/p\u003e \u003cp\u003eIn summary, our biochemical and structural analyses of Casλ2 advance our comprehension of the diverse phage-encoded type V CRISPR-Cas effectors. Phage genomes reportedly serve as natural reservoirs of miniature CRISPR-Cas effectors, which hold great promise as \u003cem\u003ein vivo\u003c/em\u003e genome editing platforms using a cargo-size-limited, single AAV vector. Therefore, our structural findings provide the molecular framework for the development of compact genome editing technologies for \u003cem\u003ein vivo\u003c/em\u003e gene therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArbor Biotechnologies is a privately funded company. 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. performed biochemical and structural analyses with help from H.M.; S.N.O., H.H., and Y.I. built the models and performed structural refinement; D.R.C. and D.A.S. designed and implemented the computational search; D.A.S., W.X.Y., and L.E.A. designed the \u003cem\u003eE. coli\u003c/em\u003e negative selection screen; L.E.A., A.O., G.M., and A.J.G. performed experimental screens and validation; S.N.O., L.E.A., Z.M. and O.N. wrote the manuscript with help from all authors; and T.D., G.R.H., Z.M., and O.N. supervised the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.M., A.J.G., A.O., L.E.A., G.R.H., T.D., W.X.Y., D.R.C., D.A.S., and Z.M. are current or former employees and shareholders of Arbor Biotechnologies. Arbor Biotechnologies has filed patents related to this work. 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\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe atomic models of Cas\u0026lambda;2\u0026ndash;crRNA\u0026ndash;target DNA ternary complexes have been deposited in the Protein Data Bank under the accession codes 9IZP (incompetent state), 9IZQ (intermediate state), 9IZR (NTS-cleaving state), and 9IZS (TS-cleaving state). The atomic model of the Cas\u0026lambda;2\u0026ndash;crRNA binary complex has been deposited in the Protein Data Bank under the accession code 9IZM. The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-61038 (incompetent state), EMD-61039 (intermediate state), EMD-61040 (NTS-cleaving state), EMD-61041 (TS-cleaving state), and EMD-61037 (binary complex).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESOURCE AVAILABILITY\u003c/strong\u003e\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\u003eMaterial availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and code availability\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eThe atomic models of Cas\u0026lambda;2\u0026ndash;crRNA\u0026ndash;target DNA ternary complexes have been deposited in the Protein Data Bank under the accession codes 9IZP (incompetent state), 9IZQ (intermediate state), 9IZR (NTS-cleaving state), and 9IZS (TS-cleaving state). The atomic model of the Cas\u0026lambda;2\u0026ndash;crRNA binary complex has been deposited in the Protein Data Bank under the accession code 9IZM. The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-61038 (incompetent state), EMD-61039 (intermediate state), EMD-61040 (NTS-cleaving state), EMD-61041 TS-cleaving state), and EMD-61037 (binary complex).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThis paper does not report original code.\u003c/li\u003e\n \u003cli\u003eAny additional information required to reanalyze the data reported in this paper is available from the Lead Contact upon request.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"METHOD DETAILS","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;negative selection screen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetagenomic data mining of run accession SRR5678926, using previously described computational discovery processes, identified an ORF associated with a CRISPR array. These elements were probed for activity using an enhanced version of the previously described \u003cem\u003eE. coli\u003c/em\u003e negative selection screen\u003csup\u003e14,15\u003c/sup\u003e, wherein RNA-programmable CRISPR-Cas interference yields spacer-specific loss of antibiotic resistance or self-targeting of essential genes, resulting in cell death. Briefly, the \u003cem\u003eE. coli\u003c/em\u003e codon-optimized genes representing the minimal CRISPR effector and accessory proteins +/- noncoding sequences were synthesized (GenScript) and cloned into a vector derived from pET-28a(+) (EMD-Millipore). The vector included an acceptor site for a CRISPR array library under the expression of the J23119 promoter. The CRISPR array library was composed of the “repeat-spacer-repeat” architecture, where “repeat’’ represents the consensus direct repeat sequence found in the CRISPR array associated with the effector, and ‘‘spacer’’ represents a variable sequence with a length determined by the mode of the spacer lengths found in the endogenous CRISPR array. The CRISPR array library containing 8,900 spacers, targeting either select \u003cem\u003eE. coli\u003c/em\u003e essential genes or regions of pACYC184, was synthesized (Twist), amplified to introduce cloning handles and unique molecular identifiers (UMIs), and then bidirectionally cloned into the library acceptor site using Golden Gate assembly.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe assembled plasmid library was electroporated into custom \u003cem\u003eE. coli\u003c/em\u003e EXPRESS BL21 (DE3) electrocompetent cells (Lucigen) harboring the pACYC184 plasmid, encapsulated in droplets using a microfluidic device manufactured in-house, and grown under antibiotic selection at 37°C with shaking. After approximately 16h, the cells were isolated from the droplets, plasmid DNA was extracted using a QIAprep Spin Miniprep kit (Qiagen), and total RNA was extracted using a custom procedure based on the Agencourt RNAdvance Tissue Kit (Beckman Coulter).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePCR was conducted on the plasmid DNA library (pre-transformation and post-harvest) using custom primers that amplify the CRISPR array and simultaneously introduce barcodes and sequencing adapters compatible with Illumina chemistry. The amplicons were pooled and then sequenced on a NextSeq 550 sequencer (Illumina). The NGS data were analyzed as described previously to determine interference activity, tracrRNA requirement, and PAM specificity\u003csup\u003e14,15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein and RNA preparation for biochemical and structural analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene encoding full-length Casλ2 (residues 1–741) was codon optimized, synthesized (Eurofins Genomics), and cloned into a modified pE-SUMO vector (LifeSensors). The mutations were introduced by a PCR-based method, using the vector encoding full-length Casλ2 as the template, and the sequences were confirmed by DNA sequencing (Supplementary Table S3). The Casλ2 proteins were expressed and purified using the protocol reported previously\u003csup\u003e13,20,25\u003c/sup\u003e. Briefly, the N-terminally His\u003csub\u003e6\u003c/sub\u003e-tagged Casλ2 proteins were expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e Rosetta2 (DE3). Transformed \u003cem\u003eE. coli\u003c/em\u003e cells were cultured at 37°C 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 β-D-thiogalactopyranoside (Nacalai Tesque). \u003cem\u003eE. coli\u003c/em\u003e cells were further cultured at 20°C overnight and harvested by centrifugation. The cells were then resuspended in buffer A (20 mM HEPES-NaOH, pH 7.6, 20 mM imidazole, and 1 M NaCl), lysed by sonication, and centrifuged. The supernatant was mixed with 3 ml Ni-NTA Superflow resin (QIAGEN), and the mixture was loaded into an Econo-Column (Bio-Rad). Proteins were eluted with buffer B (20 mM HEPES-NaOH, pH 7.6, 0.3 M imidazole, 0.3 M NaCl) and then loaded onto a 5-ml HiTrap Heparin HP column (GE Healthcare) equilibrated with buffer C (20 mM HEPES-NaOH, pH 7.6, and 0.3 M NaCl). The proteins were eluted with a linear gradient of 0.5–2 M NaCl. The purified proteins were stored at −80°C until use. The crRNAs for structural analysis were transcribed\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e with T7 RNA polymerase and purified by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis.\u0026nbsp;The crRNAs for biochemical analysis were synthesized (Ajinomoto Bio-Pharma).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;DNA cleavage experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA cleavage activity of Casλ2 was measured by \u003cem\u003ein vitro\u003c/em\u003e DNA cleavage experiments. First, these experiments were performed with the purified Casλ2, the crRNA containing a 20-nt spacer, and linearized pUC119 plasmids (100 ng, 4.7 nM) containing the target sequence with the TTN PAM. Next, the cleavage activities of Casλ2 were measured, using the linearized pUC119 plasmid with the TTA PAM. The linearized pUC119 plasmid (100 ng, 4.7 nM) was incubated at 37°C (25–75°C for Fig. 1g) for 5–60 min with the Casλ2–crRNA complex (750 nM) in 20 μL of reaction buffer, containing 20 mM HEPES-NaOH, pH 7.6, 50 mM NaCl (25–200 mM NaCl for Fig. 1f), 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT, and 2% 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\n\u003cp\u003e\u003cstrong\u003eElectron microscopy sample preparation and data collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Casλ2–crRNA–target DNA ternary complex was reconstituted by mixing purified Casλ2, the 56-nt crRNA, the 40-nt target DNA, and the 40-nucleotide non-target DNA at a molar ratio of 1:1.2:1.4:1.4. Each DNA strand has phosphorothioate modifications within the phosphate backbone around the cleavage site to inhibit DNA hydrolysis. The Casλ2–crRNA–target DNA ternary complex in the TS-cleaving state was reconstituted by mixing purified Casλ2, the 56-nt crRNA, the 40-nt target DNA, and the 14-nucleotide non-target DNA at a molar ratio of 1:1.2:1.4:1.4, with the target DNA strand bearing phosphorothioate modifications within the phosphate backbone around the cleavage site. The Casλ2 (D324A)–pre-crRNA binary complex was reconstituted by mixing purified Casλ2 and the 76-nt pre-crRNA at a molar ratio of 1:1.2. The Casλ2–crRNA–target DNA ternary complexes and the Casλ2 (D324A)–pre-crRNA binary complex were purified by size-exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated with buffer D (20 mM HEPES-NaOH, pH 7.6, 50 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 μM ZnCl\u003csub\u003e2\u003c/sub\u003e, and 1 mM DTT). The purified complex solutions (A\u003csub\u003e260\u0026nbsp;nm\u003c/sub\u003e = 3 for the ternary complexes and A\u003csub\u003e260\u0026nbsp;nm\u003c/sub\u003e = 2 for the binary complex) were then applied to Au 300-mesh R1.2/1.3 grids (Quantifoil) that were glow-discharged after adding 3 μL amylamine 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\u0026nbsp;and\u0026nbsp;cooled to the temperature of liquid nitrogen.\u003c/p\u003e\n\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 of the Casλ2–crRNA–target DNA ternary complexes were collected with a total dose of approximately 50 electrons per Å\u003csup\u003e2\u003c/sup\u003e per 48 frames by the standard mode, and datasets of the Casλ2–pre-crRNA binary complex were collected with a total dose of approximately 50 electrons per Å\u003csup\u003e2\u003c/sup\u003e per 64 frames by the CDS mode, using the EPU software (Thermo Fisher Scientific). The dose-fractionated movies were subjected to beam-induced motion correction and dose weighting using MotionCor2 implemented in RELION-3.1, and the contrast transfer function (CTF) parameters were estimated using Patch-based CTF estimation in cryoSPARC v3.3.2\u003csup\u003e31,32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle-particle cryo-EM data processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were processed using cryoSPARC v3.3.2 and RELION-3.1\u003csup\u003e32,33\u003c/sup\u003e. For the Casλ2–crRNA–target DNA ternary complex, 4,084,635 particles were initially picked from the 3,087 motion-corrected and dose-weighted micrographs using Template picker, and extracted at a pixel size of 3.32 Å in the cryoSPARC platform. These particles were subjected to several rounds of cryoSPARC 2D classification to curate particle sets. The particles were further curated by heterogeneous refinement, using maps derived from \u003cem\u003eab initio\u003c/em\u003e reconstruction as templates. The selected particles were subjected to 3D variability analysis and the maps with different conformations were used for subsequent heterogeneous refinement, resulting in the reconstructions of three distinct conformational states\u0026nbsp;(State I, the catalytically incompetent state; State II, the intermediate state; State III, the NTS-cleaving state)\u003csup\u003e34\u003c/sup\u003e.\u0026nbsp;Particles corresponding to each state were imported into RELION and subjected to 3D classification without alignment. The particle sets with the most detailed features after 3D classification were subjected to Bayesian polishing\u003csup\u003e35\u003c/sup\u003e. The resulting particles were then imported back to cryoSPARC, and Non-uniform refinement yielded maps at 2.89\u0026nbsp;Å (State I, the catalytically incompetent state), 3.06 Å (State II, the intermediate state), and 2.93 Å (State III, the NTS-cleaving state) resolutions, according to the Fourier shell correlation (FSC) criterion of 0.143\u003csup\u003e36,37\u003c/sup\u003e. The local resolution was estimated by BlocRes in cryoSPARC.\u003c/p\u003e\n\u003cp\u003eThe datasets for the Casλ2–crRNA–target DNA ternary complex in the TS-cleaving state and the Casλ2–pre-crRNA binary complex were processed using cryoSPARC and RELION in similar manners. For data processing details, see Extended Data Fig. 1 (the catalytically incompetent state, the intermediate state, and the NTS-cleaving state), 6 (the TS-cleaving state), and 7 (the binary complex).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building and validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe models were built using the protein model derived from the SWISS-MODEL server as the reference, followed by manual model building with Coot\u003csup\u003e38–42\u003c/sup\u003e. The models were refined using phenix.real_space_refine version 1.20.1, with secondary structure and metal coordination restraints\u003csup\u003e43\u003c/sup\u003e. The metal coordination restraints were generated using ReadySet, as implemented in PHENIX. The stereochemical restraints for phosphorothioate modified DNA links were generated manually. The models were validated using MolProbity\u003csup\u003e44\u003c/sup\u003e. Molecular graphics figures were prepared with UCSF ChimeraX-1.7.1\u003csup\u003e45\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;pre-crRNA processing experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pre-crRNAs were transcribed\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e with T7 RNA polymerase and purified by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis. The purified Casλ2 proteins and the pre-crRNAs were mixed at a molar ratio of 2:1 in 20 μL of reaction buffer, containing 20 mM HEPES-NaOH, pH 7.6, 50 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT, and 2% glycerol, and then incubated at 37°C for 30 min. The reactions were stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng), and then analyzed by 10% denaturing (7 M urea) polyacrylamide gel electrophoresis. The gels were stained with SYBR Gold (Invitrogen). \u003cem\u003eIn vitro\u003c/em\u003e processing experiments were performed at least three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQUANTIFICATION AND STATISTICAL ANALYSIS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are expressed as mean ± SD. 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. Comparisons between the two groups were analyzed using the two- sided unpaired t-test. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHille, F. \u003cem\u003eet al.\u003c/em\u003e The Biology of CRISPR-Cas: Backward and Forward. \u003cem\u003eCell\u003c/em\u003e\u003cstrong\u003e172\u003c/strong\u003e, 1239\u0026ndash;1259 (2018).\u003c/li\u003e\n\u003cli\u003eMakarova, K. 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F. \u003cem\u003eet al.\u003c/em\u003e UCSF ChimeraX: Structure visualization for researchers, educators, and developers. \u003cem\u003eProtein Sci.\u003c/em\u003e\u003cstrong\u003e30\u003c/strong\u003e, 70\u0026ndash;82 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5481685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5481685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ, a recently identified miniature type V effector encoded in phage genomes, has emerged as a promising candidate for genome editing due to its nuclease activity in mammalian and plant cells. However, the detailed molecular mechanisms of Casλ family of enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. The structures revealed that, unlike other type V CRISPR-Cas effectors, the REC2 domain directly interacts with the substrate DNA within the RuvC active site to facilitate the target DNA cleavage. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.\u003c/p\u003e","manuscriptTitle":"Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-19 12:23:42","doi":"10.21203/rs.3.rs-5481685/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5bedf02b-3edb-4238-a31d-48d00c4d5d42","owner":[],"postedDate":"March 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41113892,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":41113893,"name":"Biological sciences/Molecular biology/CRISPR-Cas systems/CRISPR-Cas9 genome editing"}],"tags":[],"updatedAt":"2025-06-06T07:08:56+00:00","versionOfRecord":{"articleIdentity":"rs-5481685","link":"https://doi.org/10.1038/s42003-025-08300-8","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-06-05 04:00:00","publishedOnDateReadable":"June 5th, 2025"},"versionCreatedAt":"2025-03-19 12:23:42","video":"","vorDoi":"10.1038/s42003-025-08300-8","vorDoiUrl":"https://doi.org/10.1038/s42003-025-08300-8","workflowStages":[]},"version":"v1","identity":"rs-5481685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5481685","identity":"rs-5481685","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}