In vivo genome editing with a novel Cj4Cas9

In vivo genome editing with a novel Cj4Cas9 | 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Share Download PDF Article In vivo genome editing with a novel Cj4Cas9 Daru Lu, Tianyi Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6451526/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Dec, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Natural CRISPR-Cas9 systems provide a rich resource for developing genome editing tools with diverse properties, including genome size, protospacer preference, and PAM specificity. In this study, we screened a panel of 11 Cas9 nucleases orthologous to CjCas9 using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 emerges as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5’-NNNGRY-3’). Cj4Cas9 demonstrates efficient disruption of the Tyr gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. To enhance its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings highlight the potential of Cj4Cas9 and its high-activity variants for both fundamental research and therapeutic applications. Biological sciences/Molecular biology/CRISPR-Cas systems/CRISPR-Cas9 genome editing Biological sciences/Biotechnology CRISPR Type II-C Cas9 AAV PCSK9 Tyr Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The CRISPR-Cas9 system is a versatile genome-editing platform capable of inducing genetic modifications across a wide range of organisms. This system comprises a Cas9 nuclease and a single-guide RNA (sgRNA), which together form an RNA-protein complex (RNP) 1 . Within this complex, the sgRNA directs the Cas9 nuclease to a specific DNA target (protospacer), where it introduces double-strand breaks (DSBs). These breaks are subsequently repaired by cellular repair mechanisms, leading to desired genetic changes 2 . Among the various Cas9 nucleases, Streptococcus pyogenes Cas9 (SpCas9) is the most widely used for genome editing 2 , 3 . However, SpCas9 has limitations, including inefficiency at certain genomic sites and a strict requirement for an NGG protospacer-adjacent motif (PAM) at the 3' end of the target sequence 2 , 4 . This PAM constraint restricts the applicability of SpCas9 in precision genome editing techniques such as base editing 5 , prime editing 6 , and site-specific DNA integration 7 . To address these limitations, researchers have explored the vast diversity of Cas9 orthologs available in public databases, which prefer different target sequences and PAMs 8 – 12 . One such ortholog is Campylobacter jejuni Cas9 (CjCas9), one of the most compact Cas9 nucleases identified to date, consisting of only 984 amino acids 13 . However, CjCas9 exhibits relatively low activity and requires a long NNNNRYAC PAM sequence, which limits its utility. To address these limitations, researchers have pursued two main strategies. On one hand, they have engineered CjCas9 to improve its activity. For example, Nakagawa et al. introduced L58K/D900K mutations into CjCas9, resulting in a high-activity variant named enCjCas9 14 ; Ruta et al. employed directed evolution to develop another high-activity variant, UltraCjCas9, which incorporates five mutations (L58K, E189G, F214I, S492V, D900K, and K913S) 15 . On the other hand, researchers have explored CRISPR-Cas9 tools derived from CjCas9 orthologs to recognize non-NNNNRYAC PAM sequences. For instance, Chen et al. identified Cj2Cas9 and Cj3Cas9 from CjCas9 orthologs, which recognize an N4CYA PAM 16 ; our previous work led to the development of Hsp1Cas9, Hsp2Cas9, and CcuCas9, which recognize N4RAA, N4CC, and N 4 CNA PAMs, respectively 17 . These advancements have expanded the versatility and utility of compact Cas9 nucleases for genome editing applications. In this study, we screened a total of 11 CjCas9 orthologs and identified Cj4Cas9, a nuclease that recognizes an N3GRT PAM and demonstrates high activity at multiple genomic sites. Notably, we show that Cj4Cas9 can effectively disrupt the mouse Tyrosinase (Tyr) gene ex vivo in zygotes and the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene in vivo in adult mice. We further engineered Cj4Cas9 to improve the activity. These findings highlight the potential of Cj4Cas9 and its high-activity variants as a powerful tool for genome editing in both basic research and therapeutic applications. Results 2.1 Investigation of CjCas9 ortholog activity We utilized the CjCas9 sequence to identify related orthologs from the NCBI Gene database. We selected 11 CjCas9 orthologs with amino acid identities ranging from 44.08% to 92.90% for further characterization (Table 1). Phylogenetic analysis revealed that two newly identified orthologs, Cj4Cas9 and Cj5Cas9, clustered closely with the previously characterized CjCas9, indicating a strong evolutionary relationship (Figure S1). In contrast, the remaining nine Cas9 orthologs formed distinct clusters separate from CjCas9. Notably, these CjCas9 orthologs exhibited variations in two or three residues corresponding to CjCas9 residues Arg866, Thr913, Ser915, and Ser951 (Figure S2). These residues are located in the PAM-interacting (PI) domain of CjCas9, and are critical for PAM recognition, as revealed by its crystal structure 18 . This suggests that these orthologs may recognize different PAM sequences. Table1 CjCas9 orthologs selected from the NCBI database Name NCBI ID Host Strain Length (aa) Identity to CjCas9 (%) Cj4Cas9 EFC33367.1 Campylobacter jejuni subsp. jejuni 414 985 89.04 Cj5Cas9 EAK9986011.1 Campylobacter jejuni strain. D7366 987 92.90 HfeCas9 WP_023947173.1 Helicobacter fennelliae MRY12-0050 1,046 51.39 Hsp3Cas9 WP_034327908.1 Helicobacter sp. MIT 03-1616 1,024 54.94 Hsp4Cas9 WP_095627812.1 Helicobacter sp. TUL 1,028 51.43 HeqCas9 WP_115570828.1 Helicobacter equorum 1,031 51.58 HjaCas9 WP_201274299.1 Helicobacter japonicus 1,025 53.77 Hpu1Cas9 WP_158654087.1 Helicobacter pullorum 1,049 51.50 Hpu2Cas9 WP_166666933.1 Helicobacter pullorum 1,050 51.40 Hpu3Cas9 WP_179853575.1 Helicobacter pullorum 1,034 52.76 We further analyzed the genomic architecture of these CRISPR-Cas9 systems. Among the orthologs, 10 exhibited a conserved arrangement, with the Cas9 ortholog followed by Cas1, Cas2, and a CRISPR array (Figure S3). The number of spacers within these arrays varied significantly, ranging from 4 to 40. Interestingly, Cco2Cas9 lacked the Cas1 and Cas2 genes, which may lost or moved to other genomic loci during evolution. Alignment of repeat and tracrRNA sequences revealed a moderate level of conservation across the orthologs (Figure S4). To assess the activity of these orthologs, we employed a previously developed GFP-activation assay 8 . In this system, a protospacer with a 7 bp random downstream sequence is inserted between the ATG start codon and the GFP coding sequence, disrupting GFP expression. The reporter construct is integrated into HEK293T cells via lentiviral transduction. If a Cas9 ortholog is functional, it will induce insertions or deletions (indels) at the target site, restoring GFP expression in a subset of cells (Figure 1A-B). We synthesized each CjCas9 ortholog with human codon optimization, cloned them into mammalian expression plasmids, and co-transfected them with the corresponding sgRNA constructs into HEK293T cells. SpCas9 was included as a positive control. Five days post-transfection, GFP-positive cells were observed for seven out of the 11 CjCas9 orthologs, demonstrating their genome-editing activity (Figure 1C). These results underscore the potential utility of CjCas9 orthologs for genome-editing applications. 2.2 Analysis of CjCas9 ortholog PAM preferences To determine the PAM preferences of the CjCas9 orthologs, GFP-positive cells were sorted using flow cytometry. The target sequences from these cells were then PCR-amplified and analyzed by deep sequencing. Using the sequencing data, we constructed PAM logos and PAM wheels to visualize the PAM preferences of each ortholog (Figures 2A and 2B). As anticipated, SpCas9 exhibited a strong preference for the canonical NGG PAM, consistent with prior studies 1,3 , thereby validating the reliability of our experimental system. Comparative analysis of the PAM profiles across the CjCas9 orthologs revealed significant differences in their sequence preferences, reflecting evolutionary adaptations unique to each variant. Among the orthologs, Cj4Cas9 stood out due to its recognition of a unique NNNGRY PAM and its ability to induce a higher proportion of GFP-positive cells compared to the others. We selected Cj4Cas9 for further investigation in subsequent studies. 2.3 Optimization of Cj4Cas9 for genome editing To evaluate whether Cj4Cas9 can perform genome editing using its own single guide RNA (sgRNA) scaffold, we designed a custom sgRNA scaffold for Cj4Cas9. This scaffold was created by fusing the 3′ end of a direct repeat with the 5′ end of the tracrRNA through a 4-nucleotide (nt) linker. Sequence alignment revealed that the CjCas9 sgRNA scaffold and the Cj4Cas9 sgRNA scaffold differed by only a single mismatch (Figures S5A). Additionally, both scaffolds formed similar secondary RNA structures (Figures S5B), suggesting functional compatibility. We then transfected HEK293T cells with Cj4Cas9 and its sgRNA scaffold, using the CjCas9 sgRNA scaffold as a control, to edit the AAVS1-TS1 locus. The results demonstrated that the Cj4Cas9 sgRNA scaffold exhibited activity comparable to that of the CjCas9 sgRNA scaffold (Figure S5C). Based on these findings, we proceeded with the CjCas9 sgRNA scaffold for subsequent experiments. Next, we investigated the optimal spacer length for Cj4Cas9-mediated genome editing. We designed ten spacers ranging from 18 to 25-nt targeting the AAVS1 site. Targeted deep sequencing revealed that spacers of 22- to 25-nt achieved higher editing efficiency compared to shorter spacers (Figure S6A). Consequently, we selected the 22-nt spacer for further studies. Next, we determined the optimal editing time for Cj4Cas9 in cells. HEK293T cells were transfected with Cj4Cas9 and its corresponding sgRNA, and samples were collected at 3, 5, and 7 days post-transfection. Targeted deep sequencing results indicated that Cj4Cas9-mediated gene editing became detectable by day 5 and remained stable thereafter (Figure S6B). In summary, these optimization experiments demonstrate that Cj4Cas9 is a functional genome-editing tool. 2.4 Test of Cj4Cas9 specificity Next, we tested the specificity of Cj4Cas9. We designed a panel of 11 sgRNAs with dinucleotide mismatches to target the AAVS1-TS1 locus. Five days after co-transfection of Cj4Cas9 with individual sgRNAs, targeted deep sequencing was performed to detect indel rates. The results showed that Cj4Cas9 displayed moderate off-target effects with sgRNAs MS2 and MS4, and minimal off-target effects with other sgRNAs (Figure S7A, upper panel). This low tolerance for mismatches ensures high specificity for precise genome-editing applications. Next, we evaluated the genome-wide off-target effects of Cj4Cas9 at the EMX1-TS10 locus using GUIDE-seq technology 19 . After transfecting cells with the Cj4Cas9 plasmid, sgRNA, and GUIDE-seq oligonucleotides, we prepared sequencing libraries for deep sequencing. Analysis of the sequencing data demonstrated efficient on-target cleavage by Cj4Cas9, as evidenced by the high GUIDE-seq read counts at the target site (Figure S7B). Notably, only one off-target site was detected with this specific sgRNA. These results highlight the high specificity of Cj4Cas9. 2.5 Cj4Cas9 enables genome editing at endogenous sites To further test the genome editing capability of Cj4Cas9, we selected a panel of 10 endogenous sites with NNNGRY PAMs targeting the AAVSI locus in HEK293T cells. Five days after the co-transfection of CjCas9 and sgRNA plasmids (Figure 3A-B), cells were harvested, and genomic DNA was extracted for target deep sequencing. Cj4Cas9 achieved robust editing efficiencies, with indel rates up to 32.81% (Figure 3C). Meanwhile, we selected a panel of seven endogenous sites with NNNGRY PAMs targeting the rosa26 locus in mouse neuroblastoma (N2a) cells. Cj4Cas9 achieved indel rates up to 29.18% (Figure 3D). These results underscore Cj4Cas9 a promising tool for genome editing in diverse cellular contexts. 2.6 Cj4Cas9 enables genome editing in zygotes To evaluate the potential of Cj4Cas9 for genome editing in mouse zygotes, we conducted experiments targeting the Tyr gene. We designed five Tyr -targeting sgRNAs and tested their activity in N2a cells (Figure 4A). Among these, Tyr -TS3 demonstrated the highest editing efficiency and was selected for subsequent experiments (Figure 4B). Previous studies have shown that bi-allelic inactivation of Tyr disrupts melanin production, resulting in albino pups 11,20 . To achieve this, Cj4Cas9 mRNA, sgRNA targeting the Tyr gene, and GFP mRNA were co-microinjected into pronuclear-stage embryos. The GFP expression was observed 24 hours after injection, indicating successful mRNA delivery (Figure S8A). Next, we optimized the concentrations of Cj4Cas9 mRNA and sgRNA for microinjection by testing three combinations: 25/10, 50/25, and 100/50 ng/µl. The 25/10 ng/µl group showed the highest blastocyst development rate, with four out of 10 injected zygotes developing into blastocysts (Figures S8B-C). In contrast, the 100/50 ng/µl group showed no blastocyst development. Both the 25/10 ng/µl and 50/25 ng/µl groups exhibited similar editing efficiencies in blastocysts (Figure S8D). Zygotes that successfully developed to the two-cell stage were transferred into surrogate mothers. Analysis of the resulting pups' coat colour confirmed successful Tyr gene editing, as evidenced by the absence of black pigmentation in the edited mice (Figure 4D). Targeted deep sequencing revealed an editing efficiency of 98.83% in an albino mouse (Figure 4E). These results demonstrate that Cj4Cas9 is an effective tool for genome editing in mouse zygotes. 2.7 Cj4Cas9 enables in vivo genome editing Next, we evaluated the potential of Cj4Cas9 for in vivo genome editing. The compact size of Cj4Cas9 enabled the co-packaging of both a cytomegalovirus (CMV)-driven Cj4Cas9 expression cassette and a U6-driven sgRNA into a single AAV vector (Figure 5A). To assess Cj4Cas9 activity in vivo, we targeted Pcsk9 , a therapeutically relevant gene involved in cholesterol homeostasis 21 . Pcsk9 encodes a protein that regulates the degradation of LDL receptors, thereby influencing the levels of low-density lipoprotein cholesterol (LDL-C), a major carrier of cholesterol (CHO) in the blood. Elevated LDL-C levels are associated with an increased risk of cardiovascular diseases, whereas reduced Pcsk9 activity has been shown to lower LDL-C and total cholesterol levels, offering protective benefits against cardiovascular conditions 22,23 . We designed five Pcsk9 -targeting sgRNAs and confirmed their activity in N2a cells (Figure S9A). All five sgRNAs exhibited comparable editing efficiencies (Figure S9B). Based on previous studies indicating that using multiple sgRNAs can enhance editing efficiency 24,25 , we packaged each sgRNA individually with Cj4Cas9 into a hepatotropic high-expression AAV serotype, AAV8 26 . The AAV-Cj4Cas9 constructs targeting Pcsk9 were mixed at equal titers and administered via tail vein injection into mice at a dose of 2 × 10 11 genome copies (GCs). A control group received PBS. Serum samples were collected at 0, 14, 28, and 42 days post-injection for analysis, and all mice were euthanized at 42 days (Figure 5B). The results revealed a 42.68% reduction in serum LDL-C levels and a 34.04% reduction in serum cholesterol in the AAV-Cj4Cas9-treated group, whereas PBS-treated mice maintained normal LDL-C and cholesterol levels at 42 days post-injection (Figure 5C-5D). Targeted deep sequencing demonstrated an average editing efficiency of 24.43% (Figure 5E). Consistent with these findings, western blot analysis confirmed a reduction in Pcsk9 protein levels in Cj4Cas9-treated mice compared to PBS-treated controls (Figure 5F). These results collectively demonstrate the efficacy of Cj4Cas9 for in vivo genome editing and its potential therapeutic application in modulating cholesterol metabolism. 2.8 Engineering of Cj4Cas9 for enhanced activity To improve the editing efficiency, we engineered Cj4Cas9 based on previously developed high-activity enCjCas9 14 and ultraCjCas9 15 . They contain mutations that enhance interactions between CjCas9 and the nucleic acids and thereby improve the DNA cleavage activity 14,15,27 . We aligned CjCas9 to enCjCas9 and ultraCjCas9 and identified corresponding mutations (Figure S10). We introduced L58K/D900K mutations into Cj4Cas9 to generate enCj4Cas9. We introduced E189G/F214I/S492V/K913S mutations identified from ultraCjCas9 into enCj4Cas9 to generate ultraCj4Cas9 (Figure 6A). The editing efficiencies of wild-type Cj4Cas9 and its engineered variants, enCj4Cas9 and ultraCj4Cas9, were systematically evaluated across 16 target sites. Targeted deep sequencing revealed that both enCj4Cas9 and ultraCj4Cas9 demonstrated significantly enhanced editing efficiencies compared to the wild-type enzyme (Figure 6B). Specifically, enCj4Cas9 and ultraCj4Cas9 exhibited 2.03-fold and 2.14-fold higher overall activity, respectively, relative to the wild-type (Figure 6C). Western blot analysis confirmed that the protein expression levels of all three variants were comparable, eliminating the possibility that increased activity was due to differential expression (Figure 6D). These results highlight the superior performance of the engineered Cj4Cas9 variants in genome editing applications. We focused on enCj4Cas9 in subsequent studies. Next, we compared the editing activity of WT Cj4Cas9, enCj4Cas9, and SpRY, a previously engineered SpCas9 variant with relaxed PAM specificity 28 . We designed a panel of 13 endogenous sites containing NNNGRY PAMs. Five days post-transfection, genomic DNA was extracted and subjected to targeted deep sequencing. The results revealed that Cj4Cas9 exhibited editing activity comparable to that of SpRY across the tested sites (Figure 7A-B). To ensure consistency, all three Cas9 orthologs were cloned into identical construct backbones, and Western blot analysis confirmed comparable protein expression levels among Cj4Cas9, enCj4Cas9, and SpRY (Figure 7C). The PAM preference of the enCj4Cas9 variant was further characterized using the GFP-activation assay. Intriguingly, the results demonstrated that enCj4Cas9 preferentially recognized a simple NNNGG PAM (Figure 7D-E). Additionally, we investigated the specificity of enCj4Cas9 by designing sgRNAs with dinucleotide mismatches targeting the AAVS1-TS1 locus. The results indicated that Cj4Cas9 and enCj4Cas9 exhibited similar specificity profiles (Figure S7A, lower panel). To assess genome-wide off-target effects, we performed the GUIDE-seq assay using a sgRNA targeting the EMX1-TS10 locus. Five days after co-transfecting HEK293T cells with Cj4Cas9/sgRNA-expressing plasmids and GUIDE-seq oligos, genomic DNA was extracted and prepared for deep sequencing. We identified an off-target site identical to the Cj4Cas9 off-target site (Figure S7B). In summary, enCj4Cas9 demonstrated specificity comparable to that of Cj4Cas9. 2.9 Enhanced Cj4Cas9 activity by fusion to non-specific DNA-binding proteins In parallel, to explore additional strategies for enhancing activity, we constructed fusion proteins by linking non-sequence-specific DNA-binding domains (DBDs), Sso7d and HMG-D, to Cj4Cas9 (Figure S11A), as previously described by Li et al. 23 . Although these DBDs improved editing activity at certain loci, neither fusion variant showed a significant overall enhancement in editing efficiency (Figure S11B-C). Western blot analysis confirmed comparable protein expression levels among the three variants, ruling out expression differences as the cause of increased activity (Figure S11D). These findings suggest that the intrinsic structural modifications in enCj4Cas9 and ultraCj4Cas9 play a more pivotal role in enhancing activity compared to fusion strategies involving DBDs. Discussion Among the numerous CRISPR-Cas9 tools developed to date, CjCas9 stands out due to its exceptionally compact size (984 amino acids), which is smaller than other Cas9 orthologs. To expand its targeting scope, we and others have previously identified five closely related CjCas9 orthologs that exhibit activity in human cells. For instance, Cj2Cas9 and Cj3Cas9 recognize an N4CYA PAM 16 , while Hsp1Cas9, Hsp2Cas9, and CcuCas9 recognize N4RAA, N4CC, and N4CNA PAMs, respectively 17 . In this study, we screened a panel of 11 CjCas9 orthologs and identified seven active nucleases, which displayed highly diverse PAM preferences, consistent with our earlier findings 29 . These orthologs represent a valuable resource for future genetic engineering efforts aimed at developing novel genome-editing tools with expanded capabilities. Among these orthologs, Cj4Cas9 is particularly noteworthy due to its compact size (985 amino acids) and unique PAM preference (NNNGRY). Its small genome size makes it highly suitable for AAV delivery, a critical advantage for therapeutic applications. Moreover, Cj4Cas9 demonstrated robust editing efficiency in both in vitro and in vivo settings, including the successful disruption of the Tyr gene in mouse zygotes and the Pcsk9 gene in adult mice. To further enhance its utility, we engineered a high-activity variant, enCj4Cas9, by introducing L58K/D900K mutations, which resulted in a two-fold increase in nuclease activity compared to the wild-type enzyme. Notably, enCj4Cas9 exhibited a simplified PAM preference (NNNGG), significantly broadening its targeting scope. These advancements highlight the potential of Cj4Cas9 and its engineered variants for more efficient genome-editing applications in both research and therapy. Conclusion In summary, we developed a novel small Cj4Cas9 that can efficiently induce genome editing in vivo. We further generated enCj4Cas9 with improved activity and a larger targeting scope. Cj4Cas9 and enCj4Cas9 are promising tools for basic research and therapeutic applications. Materials and Methods 3.1 Animals Age-matched C57BL/6 and ICR mice (Institute of Laboratory Animal Sciences, JSJ, China) were used as controls. All experimental mice were maintained in the animal facility of School of Life Sciences, Fudan University. Mice were housed in a 12-h light/dark cycle, with enough water and food. The mouse procedures were approved by the Institutional Animal Care and Use Committee of Fudan University (No.: 2024JS075). All procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. 3.2 Cas9 expression plasmid construction The plasmid Cas9-AAV was amplified by the primers Cas9-F/Cas9-R to obtain the Cas9-AAV backbone. The human codon optimized Cas9 gene (Supplementary file 1) was synthesized by HuaGene (Shanghai, China) and cloned into the Cas9-AAV backbone by the NEBuilder assembly tool (NEB) according to the manufacturer’s instructions. Sequences of Cas9 were confirmed by Sanger sequencing (Azenta, Suzhou, China). 3.3 sgRNA expression plasmid construction The sgRNA expression plasmids were constructed by ligating sgRNA into the Bbs I-digested U6-Cj_scaffold plasmid，which is the same as CjCas9-scaffold. The primer sequences and target sequences are listed in Supplementary file 2 and Supplementary file 3, respectively. 3.4 Cell culture and transfection The cell culture reagents were purchased from Gibco unless otherwise indicated. HEK293T and N2a cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM). All cell cultures were supplemented with 10% fetal bovine serum (FBS) (Gibco) that was inactivated at 56 °C for 30 min and 1% penicillin-streptomycin (Gibco). All cells were cultured in a humidified incubator at 37 °C and 5% CO 2 . All cell line identities were validated by STR profiling (ATCC) and repeatedly tested for mycoplasma by PCR. HEK293T and N2a cells were transfected with Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. For transient transfection, a total of 500 ng Cas9-expressing plasmid and 300 ng sgRNA plasmid were co-transfected into a 24-well plate. For Cas9 PAM sequence screening, 1.2×10 7 HEK293T cells were transfected with 10 μg of Cas9 plasmid and 5 μg of sgRNA plasmid in 10 cm dishes. 3.5 PAM discovery assay The PAM discovery assays were performed essentially as previously described, using a library of 7N. Transfected library cells with a certain percentage of GFP-positive cells were collected by centrifugation at 1000 rpm for 5 min and resuspended in PBS. Then, GFP-positive cells were collected by flow cytometry and cultured in six-well plates. Five days after culture, we extracted the genome and built deep sequencing library. 3.6 Genome editing and deep sequencing analysis of indels for endogenous sites Cells were seeded into 24-well plates one day prior to transfection and transfected at 70–80% confluency using Lipofectamine 2000 (Life Technologies) following the manufacturer’s recommended protocol. For genome editing, 10 6 cells were transfected with a total of 500 ng of Cas9 plasmid and 300 ng of sgRNA plasmid in 28-well plates. Six days after transfection, the cells were harvested, and genomic DNA was extracted in QuickExtract DNA Extraction Solution (Epicenter). To measure indel frequencies, the target sites were amplified by two rounds of nested PCR to add the Illumina adaptor sequence. The PCR products (200-300 bp in length) were gel-extracted by a Gel Extraction Kit (TIANGEN) for deep sequencing. 3.7 mRNA and gRNA preparation The Cj4Cas9 and GFP were PCR amplified with primers containing T7 promoter and 75 nt poly-A tail with KOD FX (TOYOBO) and transcribed in vitro using the RNA transcription kit (novoprotein). Cj4Cas9 gRNA was PCR amplified with primers containing T7 promoter. These in vitro transcribed mRNA and gRNA were stored at -80°C after purification with Monarch RNA cleanup kit (NEB) according to the manufacture’s protocol. Aliquoting was necessary for convenience and longer storage. 3.8 Microinjection of mouse zygotes and genotyping Groups of 30 pronuclear-stage zygotes were injected with Cj4Cas9 mRNA (25 ng/µL) and sgRNA (12.5 ng/µL). The zygotes were cultured in KSOM at 37 °C with 5% CO 2 . The zygotes were collected individually with QuickExtract DNA Extraction Solution (Epicenter) when developed into blastocyst stage to analyze gene editing efficiencies. Embryo lysis were subjected to PCR amplification. Amplicons containing target sequences were analyzed by deep sequencing. 3.9 GUIDE-seq Off-target assay We performed a GUIDE-seq experiment with some modifications to the original protocol, as described 19 . On the day of the experiment, 2x10 5 HEK293T cells per target site were harvested and washed in PBS and transfected with 500 ng of Cas9 plasmid, 500 ng of sgRNA plasmid and 100 pmol annealed GUIDE-seq oligonucleotides through the Neon Transfection System. The electroporation voltage, width, and number of pulses were 1150 V, 20 ms, and 2 pulses, respectively. Genomic DNA was extracted with the DNeasy Blood and Tissue kit (QIAGEN) for 6-10 days according to cell proliferation after electroporation according to the manufacturer’s protocol. The genome library was prepared and subjected to deep sequencing. 3.10 Histology and Serum Analysis Tissues were fixed using 4% PFA at 4 °C overnight and dehydrated the next day before paraffinization. Paraffin blocks were cut into 5 µm thick sections, deparaffinized with xylene, and rehydrated. Sections were stained for DAPI and examined for transduction efficacy. Serum levels of LDL-C were evaluated by surfactant removal method (Gcell) following the manufacturer's instructions. Similarly, the Total Cholesterol Assay Kit (Gcell) was utilized to measure CHO levels in the serum. 3.11 Statistics All statistics used in this study were performed on at least n = 3 biologically independent experiments and calculated using an unpaired or paired two-tailed Student's t-test using R package dplyr. Detailed information on samples and experimental replicates can be found in the figure legends. p values less than 0.05 were considered significant, denoted as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Declarations Author Contributions: TY. Wang conceived and designed the experiments. TY. Wang, YF. Tian, Y. Zhang, MR. Li, J. Luo, and BW. Chen performed the experiments. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by grants from the National Key Research and Development Program of China (2023YFC2705600, 2023YFC2705602, 2021YFA0910602); the National Natural Science Foundation of China (82370254, 82070258); and the Science and Technology Research Program of Shanghai (24HC2810100, 23ZR1426000). Institutional Review Board Statement: Informed Consent Statement: Informed consents were obtained from all subjects involved in the study. Data Availability Statement:  Conflicts of Interest: The authors declare no conflict of interest. References Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337 , 816-821 (2012). https://doi.org:10.1126/science.1225829 Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8 , 2281-2308 (2013). https://doi.org:10.1038/nprot.2013.143 Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. 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Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range. Commun Biol 5 , 211 (2022). https://doi.org:10.1038/s42003-022-03149-7 Ruta, G. V. et al. Eukaryotic-driven directed evolution of Cas9 nucleases. Genome Biol 25 , 79 (2024). https://doi.org:10.1186/s13059-024-03215-9 Chen, S. et al. Compact Cje3Cas9 for Efficient In Vivo Genome Editing and Adenine Base Editing. CRISPR J 5 , 472-486 (2022). https://doi.org:10.1089/crispr.2021.0143 Gao, S. et al. Genome editing with natural and engineered CjCas9 orthologs. Mol Ther 31 , 1177-1187 (2023). https://doi.org:10.1016/j.ymthe.2023.01.029 Yamada, M. et al. Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems. Mol Cell 65 , 1109-1121 e1103 (2017). https://doi.org:10.1016/j.molcel.2017.02.007 Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33 , 187-197 (2015). https://doi.org:10.1038/nbt.3117 Wang, M. et al. Hypercompact TnpB and truncated TnpB systems enable efficient genome editing in vitro and in vivo. Cell Discov 10 , 31 (2024). https://doi.org:10.1038/s41421-023-00645-w Musunuru, K. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593 , 429-434 (2021). https://doi.org:10.1038/s41586-021-03534-y Sabatine, M. S. et al. Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease. N Engl J Med 376 , 1713-1722 (2017). https://doi.org:10.1056/NEJMoa1615664 Yin, S. et al. Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies. Mol Ther 31 , 744-759 (2023). https://doi.org:10.1016/j.ymthe.2022.11.014 Jang, D. E. et al. Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency. Exp Mol Med 50 , 1-9 (2018). https://doi.org:10.1038/s12276-018-0037-x Li, J., Kong, D., Ke, Y., Zeng, W. & Miki, D. Application of multiple sgRNAs boosts efficiency of CRISPR/Cas9-mediated gene targeting in Arabidopsis. BMC Biol 22 , 6 (2024). https://doi.org:10.1186/s12915-024-01810-7 Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J. E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 16 , 1073-1080 (2008). https://doi.org:10.1038/mt.2008.76 Schmidheini, L. et al. Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility. Nat Chem Biol 20 , 333-343 (2024). https://doi.org:10.1038/s41589-023-01427-x Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368 , 290-296 (2020). https://doi.org:10.1126/science.aba8853 Wei, J. et al. Closely related type II-C Cas9 orthologs recognize diverse PAMs. Elife 11 (2022). https://doi.org:10.7554/eLife.77825 Additional Declarations There is NO Competing Interest. Supplementary Files Cj4Cas9Suppl.doc Supplemental Material Cite Share Download PDF Status: Published Journal Publication published 30 Dec, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6451526","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":453657914,"identity":"a1352690-556a-46af-ae82-f8f9b35589a9","order_by":0,"name":"Daru Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApUlEQVRIiWNgGAWjYDCCA0D8gMGGh5+/gRQtCQxpMpIzDpCm5bCNQUMCkTr4jp99JpHYdp7HgOEA44ePOURokTyTbgbUcpvHnLmBWXLmNiK0GBxIYwNrsWw4wMbMS5SW889AWs7xGBxIIFbLDbAtB0jQInnjGbNFwrlkHskZB5uJ8wvf+TTGGx/K7Oz5+ZsPfvhIjBYkwNhAmvpRMApGwSgYBbgBAIHwNj3D8HvnAAAAAElFTkSuQmCC","orcid":"","institution":"Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Daru","middleName":"","lastName":"Lu","suffix":""},{"id":453657915,"identity":"91ec2e05-4755-4dca-9ce1-ba0f52cc3a0c","order_by":1,"name":"Tianyi Wang","email":"","orcid":"https://orcid.org/0009-0008-3440-8617","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Tianyi","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-04-15 06:25:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6451526/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6451526/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-09430-9","type":"published","date":"2025-12-30T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82594657,"identity":"82cde135-3266-4efc-ba3b-d6a2c0f42d16","added_by":"auto","created_at":"2025-05-13 08:36:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12175269,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the ortholog of CjCas9. (A) Schematic representation of the GFP-activation assay. A 7-bp random sequence followed by a 25-bp protospacer (target sequence) was inserted between the ATG start codon and the GFP-coding sequence, disrupting GFP expression. The reporter construct was packaged into a lentivirus vector and transfected into cells. sgRNA/Cas9-expressing plasmids were subsequently transfected into the reporter cells. Successful editing of the target sequence restored GFP expression. GFP-positive cells were sorted and analyzed via PCR amplification and deep sequencing to assess the activity of CjCas9 orthologs. (B) Schematic of the GFP-activation assay designed to assess the activity of CjCas9 orthologs. The Cas9 expression plasmid and the sgRNA expression plasmid are co-transfected into reporter cells for genome editing. (C) Representative images demonstrating GFP expression induced by CjCas9 orthologs. Cells transfected with SpCas9 served as a positive control for GFP activation. BF, bright field; GFP, green fluorescent protein.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6451526/v1/e9da86ae4fcb4dffc7651f51.png"},{"id":82594640,"identity":"b6cd3769-1e26-405b-aaf1-2a4b6f0b8658","added_by":"auto","created_at":"2025-05-13 08:36:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3000159,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the PAM sequence of CjCas9 orthologs. (A) WebLogos were generated based on the deep sequencing data. (B) PAM wheels were generated based on the deep sequencing data.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6451526/v1/da3d6111f5b4b2f7056f501d.png"},{"id":82595727,"identity":"81665c7d-0f80-4016-87a7-daa048ebcd79","added_by":"auto","created_at":"2025-05-13 08:44:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2711313,"visible":true,"origin":"","legend":"\u003cp\u003eGenome editing capability of Cj4Cas9. (A) Schematic representation of the experimental design to assess Cj4Cas9 editing efficiency. (B) Illustration of the workflow for editing efficiency and mismatch tolerance assays. (C) Genome-editing efficiency of Cj4Cas9 in HEK293T cells, showing indel rates at target sites. (D) Editing efficiency of Cj4Cas9 in N2a cells, demonstrating comparable activity across mammalian cell types.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6451526/v1/3610590b0a617829c226e855.png"},{"id":82594644,"identity":"31f7cd70-3bc1-483b-aca9-6c975dc84d69","added_by":"auto","created_at":"2025-05-13 08:36:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4607412,"visible":true,"origin":"","legend":"\u003cp\u003eCj4Cas9 efficiently induces genome editing in mouse zygotes. (A) Schematic diagram of the mouse \u003cem\u003eTyrosinase\u003c/em\u003e (\u003cem\u003eTyr\u003c/em\u003e) gene. The sgRNA target loci are shown. Red lines indicate PAMs. (B) Indel frequencies of Cj4Cas9 at 5 \u003cem\u003eTyr\u003c/em\u003esites in N2a cell line (n=3). Data are presented as the mean ± SD. (C) Workflow for genome editing in mouse zygotes. (D) Photograph of mice at one week after birth. Mice with mutations in \u003cem\u003eTyr\u003c/em\u003e presented the phenotype of albino. (E) Deep sequencing results showed indels in an edited mouse.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6451526/v1/2603b415810484318bf59e62.png"},{"id":82595731,"identity":"2f4213f0-1005-4732-90bb-37fb89a4014e","added_by":"auto","created_at":"2025-05-13 08:44:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1052641,"visible":true,"origin":"","legend":"\u003cp\u003eAAV8-delivery of Cj4Cas9 for in vivo genome editing in mice. (A) Schematic diagram of the AAV8-Cj4Cas9 vectors. CMV, cytomegalovirus. U6, U6 promoter. ITR, inverted terminal repeats. (B) The procedure of in vivo genome editing in mice. Viruses are administered intravenously via tail vein injection in 4-5 weeks C57BL/6J mice. (C-D) Time course of total serum cholesterol and LDL-C in animals treated with AAV8-Cj4Cas9 respectively (n=3). (E) Indel efficiencies of \u003cem\u003ePcsk9\u003c/em\u003e in mouse liver. The PBS-injected group serves as a control (n = 3). (F) Analysis of \u003cem\u003ePcsk9\u003c/em\u003e protein expression in mouse liver by Western blot. Data represent means ± SD. ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6451526/v1/afb2ecfc99d08a725e08af3b.png"},{"id":82594662,"identity":"2f72b08c-3439-47ca-afc3-9a474d3f1bfd","added_by":"auto","created_at":"2025-05-13 08:36:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1244800,"visible":true,"origin":"","legend":"\u003cp\u003eEngineering of Cj4Cas9 for enhanced activity. (A) Schematic representation of the mutation sites introduced in enCj4Cas9 and ultraCj4Cas9, highlighting the key amino acid changes engineered to improve activity. (B) Indel efficiencies across 16 target sites, demonstrating the enhanced performance of enCj4Cas9 and ultraCj4Cas9 compared to wild-type Cj4Cas9. (C) Box plots summarizing the editing efficiencies of wild-type Cj4Cas9, enCj4Cas9, and ultraCj4Cas9, illustrating the overall increase in activity of the engineered variants. (D) Western blot analysis confirming comparable protein expression levels of Cj4Cas9, enCj4Cas9, and ultraCj4Cas9 in transfected cells. GAPDH was used as a loading control to ensure equal protein loading. Data represent mean ± SD for n=3 biologically independent experiments. p values were determined using a two-sided Student’s t-test. ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6451526/v1/d595b49c4a5597ab36c69ba1.png"},{"id":82594656,"identity":"3fc46b10-b05e-45a8-9771-fda5bedb4188","added_by":"auto","created_at":"2025-05-13 08:36:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1926405,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of enCj4Cas9 for genome editing. (A) Indel efficiencies of Cj4Cas9, enCj4Cas9, and SpRY at selected target sites. (B) Box plots illustrate no significant differences in editing efficiencies between enCj4Cas9 and SpRY. (C) Western blot analysis showing equivalent protein expression levels of Cj4Cas9, enCj4Cas9, and SpRY in transfected cells. (D) PAM logo and (E) PAM wheel diagrams show that enCj4Cas9 recognizes an NNNGG PAM. Data represent mean ± SD for n=3 biologically independent experiments. p values were determined using a two-sided Student’s t-test. ∗p \u0026lt; 0.05, ∗∗p \u0026lt; 0.01, ∗∗∗p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6451526/v1/aefcf2520adc2115a4dbb38f.png"},{"id":101839375,"identity":"87c751a6-515b-49ca-b77b-abe702c0f205","added_by":"auto","created_at":"2026-02-04 08:20:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26098685,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6451526/v1/927b6a96-690a-4c5a-935d-3bd2f2bfe956.pdf"},{"id":82594649,"identity":"c3f7b6ec-6328-4c63-bc18-b7837519d2d4","added_by":"auto","created_at":"2025-05-13 08:36:24","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30574592,"visible":true,"origin":"","legend":"Supplemental Material","description":"","filename":"Cj4Cas9Suppl.doc","url":"https://assets-eu.researchsquare.com/files/rs-6451526/v1/4e97848508c8b766f17660ea.doc"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"In vivo genome editing with a novel Cj4Cas9","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe CRISPR-Cas9 system is a versatile genome-editing platform capable of inducing genetic modifications across a wide range of organisms. This system comprises a Cas9 nuclease and a single-guide RNA (sgRNA), which together form an RNA-protein complex (RNP) \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Within this complex, the sgRNA directs the Cas9 nuclease to a specific DNA target (protospacer), where it introduces double-strand breaks (DSBs). These breaks are subsequently repaired by cellular repair mechanisms, leading to desired genetic changes \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong the various Cas9 nucleases, \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e Cas9 (SpCas9) is the most widely used for genome editing \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, SpCas9 has limitations, including inefficiency at certain genomic sites and a strict requirement for an NGG protospacer-adjacent motif (PAM) at the 3' end of the target sequence \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This PAM constraint restricts the applicability of SpCas9 in precision genome editing techniques such as base editing \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, prime editing \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, and site-specific DNA integration \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. To address these limitations, researchers have explored the vast diversity of Cas9 orthologs available in public databases, which prefer different target sequences and PAMs \u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne such ortholog is \u003cem\u003eCampylobacter jejuni\u003c/em\u003e Cas9 (CjCas9), one of the most compact Cas9 nucleases identified to date, consisting of only 984 amino acids \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, CjCas9 exhibits relatively low activity and requires a long NNNNRYAC PAM sequence, which limits its utility. To address these limitations, researchers have pursued two main strategies. On one hand, they have engineered CjCas9 to improve its activity. For example, Nakagawa et al. introduced L58K/D900K mutations into CjCas9, resulting in a high-activity variant named enCjCas9 \u003csup\u003e14\u003c/sup\u003e; Ruta et al. employed directed evolution to develop another high-activity variant, UltraCjCas9, which incorporates five mutations (L58K, E189G, F214I, S492V, D900K, and K913S) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. On the other hand, researchers have explored CRISPR-Cas9 tools derived from CjCas9 orthologs to recognize non-NNNNRYAC PAM sequences. For instance, Chen et al. identified Cj2Cas9 and Cj3Cas9 from CjCas9 orthologs, which recognize an N4CYA PAM \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e; our previous work led to the development of Hsp1Cas9, Hsp2Cas9, and CcuCas9, which recognize N4RAA, N4CC, and N\u003csub\u003e4\u003c/sub\u003eCNA PAMs, respectively \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These advancements have expanded the versatility and utility of compact Cas9 nucleases for genome editing applications.\u003c/p\u003e \u003cp\u003eIn this study, we screened a total of 11 CjCas9 orthologs and identified Cj4Cas9, a nuclease that recognizes an N3GRT PAM and demonstrates high activity at multiple genomic sites. Notably, we show that Cj4Cas9 can effectively disrupt the mouse \u003cem\u003eTyrosinase (Tyr)\u003c/em\u003e gene ex vivo in zygotes and the \u003cem\u003eproprotein convertase subtilisin/kexin type 9 (Pcsk9)\u003c/em\u003e gene in vivo in adult mice. We further engineered Cj4Cas9 to improve the activity. These findings highlight the potential of Cj4Cas9 and its high-activity variants as a powerful tool for genome editing in both basic research and therapeutic applications.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e2.1\u0026nbsp;Investigation of CjCas9 ortholog activity\u003c/p\u003e\n\u003cp\u003eWe utilized the CjCas9 sequence to identify related orthologs from the NCBI Gene database. We selected\u0026nbsp;11 CjCas9 orthologs\u0026nbsp;with amino acid identities ranging from\u0026nbsp;44.08% to 92.90%\u0026nbsp;for further characterization (Table 1). Phylogenetic analysis revealed that two newly identified orthologs,\u0026nbsp;Cj4Cas9\u0026nbsp;and\u0026nbsp;Cj5Cas9, clustered closely with the previously characterized CjCas9, indicating a strong evolutionary relationship (Figure S1). In contrast, the remaining\u0026nbsp;nine Cas9 orthologs\u0026nbsp;formed distinct clusters separate from CjCas9. Notably, these CjCas9 orthologs exhibited variations in two or three residues corresponding to\u0026nbsp;CjCas9 residues Arg866, Thr913, Ser915, and Ser951\u0026nbsp;(Figure S2). These residues are located in the PAM-interacting (PI) domain of CjCas9, and are critical for PAM recognition, as revealed by its crystal structure \u003csup\u003e18\u003c/sup\u003e. This suggests that these orthologs may recognize different PAM sequences.\u003c/p\u003e\n\u003cp\u003eTable1 CjCas9 orthologs selected from the NCBI database\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"576\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eNCBI ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003eHost Strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eLength (aa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003eIdentity to CjCas9 (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCj4Cas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eEFC33367.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003eCampylobacter jejuni subsp. jejuni 414\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e985\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e89.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCj5Cas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eEAK9986011.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003eCampylobacter jejuni strain. D7366\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e92.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHfeCas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003eWP_023947173.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003eHelicobacter fennelliae MRY12-0050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1,046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e51.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHsp3Cas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eWP_034327908.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 188px;\"\u003e\n \u003cp\u003eHelicobacter sp. MIT 03-1616\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1,024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003e54.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHsp4Cas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eWP_095627812.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 188px;\"\u003e\n \u003cp\u003eHelicobacter sp. TUL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1,028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003e51.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHeqCas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eWP_115570828.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 188px;\"\u003e\n \u003cp\u003eHelicobacter equorum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1,031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003e51.58\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHjaCas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eWP_201274299.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 188px;\"\u003e\n \u003cp\u003eHelicobacter japonicus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1,025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003e53.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHpu1Cas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eWP_158654087.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 188px;\"\u003e\n \u003cp\u003eHelicobacter pullorum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1,049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003e51.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHpu2Cas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eWP_166666933.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 188px;\"\u003e\n \u003cp\u003eHelicobacter pullorum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1,050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003e51.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHpu3Cas9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 142px;\"\u003e\n \u003cp\u003eWP_179853575.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 188px;\"\u003e\n \u003cp\u003eHelicobacter pullorum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e1,034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003e52.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eWe further analyzed the genomic architecture of these CRISPR-Cas9 systems. Among the orthologs, 10 exhibited a conserved arrangement, with the Cas9 ortholog followed by Cas1, Cas2, and a CRISPR array (Figure S3). The number of spacers within these arrays varied significantly, ranging from 4 to 40. Interestingly, Cco2Cas9 lacked the Cas1 and Cas2 genes, which may lost or moved to other genomic loci during evolution. Alignment of repeat and tracrRNA sequences revealed a moderate level of conservation across the orthologs (Figure S4).\u003c/p\u003e\n\u003cp\u003eTo assess the activity of these orthologs, we employed a previously developed GFP-activation assay \u003csup\u003e8\u003c/sup\u003e. In this system, a protospacer with a\u0026nbsp;7 bp random downstream sequence\u0026nbsp;is inserted between the\u0026nbsp;ATG start codon\u0026nbsp;and the\u0026nbsp;GFP coding sequence, disrupting GFP expression. The reporter construct is integrated into\u0026nbsp;HEK293T cells\u0026nbsp;via lentiviral transduction. If a Cas9 ortholog is functional, it will induce\u0026nbsp;insertions or deletions (indels)\u0026nbsp;at the target site, restoring GFP expression in a subset of cells (Figure 1A-B). We synthesized each CjCas9 ortholog with\u0026nbsp;human codon optimization, cloned them into mammalian expression plasmids, and co-transfected them with the corresponding sgRNA constructs into HEK293T cells.\u0026nbsp;SpCas9\u0026nbsp;was included as a positive control. Five days post-transfection,\u0026nbsp;GFP-positive cells\u0026nbsp;were observed for\u0026nbsp;seven out of the 11 CjCas9 orthologs, demonstrating their genome-editing activity (Figure 1C). These results underscore the potential utility of CjCas9 orthologs for genome-editing applications.\u003c/p\u003e\n\u003ch3\u003e2.2 Analysis of CjCas9 ortholog PAM preferences\u003c/h3\u003e\n\u003cp\u003eTo determine the PAM preferences of the CjCas9 orthologs, GFP-positive cells were sorted using flow cytometry. The target sequences from these cells were then PCR-amplified and analyzed by deep sequencing. Using the sequencing data, we constructed PAM logos and PAM wheels to visualize the PAM preferences of each ortholog (Figures 2A and 2B). As anticipated, SpCas9 exhibited a strong preference for the canonical NGG PAM, consistent with prior studies \u003csup\u003e1,3\u003c/sup\u003e, thereby validating the reliability of our experimental system. Comparative analysis of the PAM profiles across the CjCas9 orthologs revealed significant differences in their sequence preferences, reflecting evolutionary adaptations unique to each variant. Among the orthologs, Cj4Cas9 stood out due to its recognition of a unique NNNGRY PAM and its ability to induce a higher proportion of GFP-positive cells compared to the others. We selected Cj4Cas9 for further investigation in subsequent studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.3 Optimization of Cj4Cas9 for genome editing\u003c/p\u003e\n\u003cp\u003eTo evaluate whether\u0026nbsp;Cj4Cas9\u0026nbsp;can perform genome editing using its own single guide RNA (sgRNA) scaffold, we designed a custom sgRNA scaffold for Cj4Cas9. This scaffold was created by fusing the\u0026nbsp;3\u0026prime; end of a direct repeat\u0026nbsp;with the\u0026nbsp;5\u0026prime; end of the tracrRNA\u0026nbsp;through a\u0026nbsp;4-nucleotide (nt) linker. Sequence alignment revealed that the\u0026nbsp;CjCas9 sgRNA scaffold\u0026nbsp;and the\u0026nbsp;Cj4Cas9 sgRNA scaffold\u0026nbsp;differed by only a single mismatch\u0026nbsp;(Figures S5A). Additionally, both scaffolds formed\u0026nbsp;similar secondary RNA structures\u0026nbsp;(Figures S5B), suggesting functional compatibility. We then transfected\u0026nbsp;HEK293T cells\u0026nbsp;with Cj4Cas9 and its sgRNA scaffold, using the CjCas9 sgRNA scaffold as a control, to edit the AAVS1-TS1 locus. The results demonstrated that the\u0026nbsp;Cj4Cas9 sgRNA scaffold\u0026nbsp;exhibited activity comparable to that of the\u0026nbsp;CjCas9 sgRNA scaffold\u0026nbsp;(Figure S5C). Based on these findings, we proceeded with the\u0026nbsp;CjCas9 sgRNA scaffold\u0026nbsp;for subsequent experiments.\u003c/p\u003e\n\u003cp\u003eNext, we investigated the\u0026nbsp;optimal spacer length\u0026nbsp;for Cj4Cas9-mediated genome editing. We designed ten spacers ranging from\u0026nbsp;18 to 25-nt\u0026nbsp;targeting the\u0026nbsp;AAVS1 site. Targeted deep sequencing revealed that spacers of\u0026nbsp;22- to 25-nt\u0026nbsp;achieved higher editing efficiency compared to shorter spacers (Figure S6A). Consequently, we selected the\u0026nbsp;22-nt spacer\u0026nbsp;for further studies.\u003c/p\u003e\n\u003cp\u003eNext, we determined the optimal editing time for Cj4Cas9 in cells. HEK293T cells were transfected with Cj4Cas9 and its corresponding sgRNA, and samples were collected at 3, 5, and 7 days post-transfection. Targeted deep sequencing results indicated that Cj4Cas9-mediated gene editing became detectable by day 5 and remained stable thereafter (Figure S6B). In summary, these optimization experiments demonstrate that Cj4Cas9 is a functional genome-editing tool.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.4 Test of Cj4Cas9 specificity\u003c/p\u003e\n\u003cp\u003eNext, we tested the specificity of Cj4Cas9. We designed a panel of 11 sgRNAs with dinucleotide mismatches to target the AAVS1-TS1 locus. Five days after co-transfection of Cj4Cas9 with individual sgRNAs, targeted deep sequencing was performed to detect indel rates. The results showed that Cj4Cas9 displayed moderate off-target effects with sgRNAs MS2 and MS4, and minimal off-target effects with other sgRNAs (Figure S7A, upper panel). This low tolerance for mismatches ensures high specificity for precise genome-editing applications.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we evaluated the genome-wide off-target effects of Cj4Cas9 at the \u003cem\u003eEMX1-TS10\u003c/em\u003e locus using GUIDE-seq technology \u003csup\u003e19\u003c/sup\u003e. After transfecting cells with the Cj4Cas9 plasmid, sgRNA, and GUIDE-seq oligonucleotides, we prepared sequencing libraries for deep sequencing. Analysis of the sequencing data demonstrated efficient on-target cleavage by Cj4Cas9, as evidenced by the high GUIDE-seq read counts at the target site (Figure S7B). Notably, only one off-target site was detected with this specific sgRNA. These results highlight the high specificity of Cj4Cas9.\u003c/p\u003e\n\u003cp\u003e2.5 Cj4Cas9 enables genome editing at endogenous sites\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further test the genome editing capability of Cj4Cas9, we selected a panel of 10 endogenous sites with NNNGRY PAMs targeting the AAVSI locus in HEK293T cells. Five days after the co-transfection of CjCas9 and sgRNA plasmids (Figure 3A-B), cells were harvested, and genomic DNA was extracted for target deep sequencing. Cj4Cas9 achieved robust editing efficiencies, with indel rates up to 32.81% (Figure 3C). Meanwhile, we selected a panel of seven endogenous sites with NNNGRY PAMs targeting the rosa26 locus in mouse neuroblastoma (N2a) cells. Cj4Cas9 achieved indel rates up to 29.18% (Figure 3D). These results underscore Cj4Cas9 a promising tool for genome editing in diverse cellular contexts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.6 Cj4Cas9 enables genome editing in zygotes\u003c/p\u003e\n\u003cp\u003eTo evaluate the potential of Cj4Cas9 for genome editing in mouse zygotes, we conducted experiments targeting the \u003cem\u003eTyr\u003c/em\u003e gene. We designed five \u003cem\u003eTyr\u003c/em\u003e-targeting sgRNAs\u0026nbsp;and tested their activity in\u0026nbsp;N2a cells (Figure 4A). Among these, \u003cem\u003eTyr\u003c/em\u003e-TS3\u0026nbsp;demonstrated the highest editing efficiency and was selected for subsequent experiments (Figure 4B). Previous studies have shown that bi-allelic inactivation of \u003cem\u003eTyr\u003c/em\u003e disrupts melanin production, resulting in albino pups \u003csup\u003e11,20\u003c/sup\u003e. To achieve this, Cj4Cas9 mRNA, sgRNA targeting the \u003cem\u003eTyr\u003c/em\u003e gene, and GFP mRNA were co-microinjected into pronuclear-stage embryos. The GFP expression was observed 24 hours after injection, indicating successful mRNA delivery (Figure S8A).\u003c/p\u003e\n\u003cp\u003eNext, we optimized the concentrations of Cj4Cas9 mRNA and sgRNA for microinjection by testing three combinations: 25/10, 50/25, and 100/50 ng/\u0026micro;l. The 25/10 ng/\u0026micro;l group showed the highest blastocyst development rate, with four out of 10 injected zygotes developing into blastocysts (Figures S8B-C). In contrast, the 100/50 ng/\u0026micro;l group showed no blastocyst development. Both the 25/10 ng/\u0026micro;l and 50/25 ng/\u0026micro;l groups exhibited similar editing efficiencies in blastocysts (Figure S8D).\u003c/p\u003e\n\u003cp\u003eZygotes that successfully developed to the two-cell stage were transferred into surrogate mothers. Analysis of the resulting pups\u0026apos; coat colour confirmed successful \u003cem\u003eTyr\u003c/em\u003e gene editing, as evidenced by the absence of black pigmentation in the edited mice (Figure 4D). Targeted deep sequencing revealed an editing efficiency of\u0026nbsp;98.83%\u0026nbsp;in an albino mouse (Figure 4E). These results demonstrate that\u0026nbsp;Cj4Cas9\u0026nbsp;is an effective tool for genome editing in mouse zygotes.\u003c/p\u003e\n\u003cp\u003e2.7 Cj4Cas9 enables \u003cem\u003ein vivo\u003c/em\u003e genome editing\u003c/p\u003e\n\u003cp\u003eNext, we evaluated the potential of Cj4Cas9 for in vivo genome editing. The compact size of Cj4Cas9 enabled the co-packaging of both a cytomegalovirus (CMV)-driven Cj4Cas9 expression cassette and a U6-driven sgRNA into a single AAV vector (Figure 5A). To assess Cj4Cas9 activity in vivo, we targeted \u003cem\u003ePcsk9\u003c/em\u003e, a therapeutically relevant gene involved in cholesterol homeostasis\u003csup\u003e21\u003c/sup\u003e. \u003cem\u003ePcsk9\u003c/em\u003e encodes a protein that regulates the degradation of LDL receptors, thereby influencing the levels of low-density lipoprotein cholesterol (LDL-C), a major carrier of cholesterol (CHO) in the blood. Elevated LDL-C levels are associated with an increased risk of cardiovascular diseases, whereas reduced \u003cem\u003ePcsk9\u003c/em\u003e activity has been shown to lower LDL-C and total cholesterol levels, offering protective benefits against cardiovascular conditions\u003csup\u003e22,23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWe designed five \u003cem\u003ePcsk9\u003c/em\u003e-targeting sgRNAs and confirmed their activity in N2a cells (Figure S9A). All five sgRNAs exhibited comparable editing efficiencies (Figure S9B). Based on previous studies indicating that using multiple sgRNAs can enhance editing efficiency\u003csup\u003e24,25\u003c/sup\u003e, we packaged each sgRNA individually with Cj4Cas9 into a hepatotropic high-expression AAV serotype, AAV8\u003csup\u003e26\u003c/sup\u003e. The AAV-Cj4Cas9 constructs targeting \u003cem\u003ePcsk9\u003c/em\u003e were mixed at equal titers and administered via tail vein injection into mice at a dose of 2 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e genome copies (GCs). A control group received PBS. Serum samples were collected at 0, 14, 28, and 42 days post-injection for analysis, and all mice were euthanized at 42 days (Figure 5B).\u003c/p\u003e\n\u003cp\u003eThe results revealed a 42.68% reduction in serum LDL-C levels and a 34.04% reduction in serum cholesterol in the AAV-Cj4Cas9-treated group, whereas PBS-treated mice maintained normal LDL-C and cholesterol levels at 42 days post-injection (Figure 5C-5D). Targeted deep sequencing demonstrated an average editing efficiency of 24.43% (Figure 5E). Consistent with these findings, western blot analysis confirmed a reduction in \u003cem\u003ePcsk9\u003c/em\u003e protein levels in Cj4Cas9-treated mice compared to PBS-treated controls (Figure 5F). These results collectively demonstrate the efficacy of Cj4Cas9 for in vivo genome editing and its potential therapeutic application in modulating cholesterol metabolism.\u003c/p\u003e\n\u003cp\u003e2.8 Engineering of Cj4Cas9 for enhanced activity\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo improve the editing efficiency, we engineered Cj4Cas9 based on previously developed high-activity enCjCas9\u003csup\u003e14\u003c/sup\u003e and ultraCjCas9\u003csup\u003e15\u003c/sup\u003e. They contain mutations that enhance interactions between CjCas9 and the nucleic acids and thereby improve the DNA cleavage activity\u003csup\u003e14,15,27\u003c/sup\u003e. We aligned CjCas9 to enCjCas9 and ultraCjCas9 and identified corresponding mutations (Figure S10). We introduced L58K/D900K mutations into Cj4Cas9 to generate enCj4Cas9. We introduced E189G/F214I/S492V/K913S mutations identified from ultraCjCas9 into enCj4Cas9 to generate ultraCj4Cas9 (Figure 6A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe editing efficiencies of wild-type Cj4Cas9 and its engineered variants, enCj4Cas9 and ultraCj4Cas9, were systematically evaluated across 16 target sites. Targeted deep sequencing revealed that both enCj4Cas9 and ultraCj4Cas9 demonstrated significantly enhanced editing efficiencies compared to the wild-type enzyme (Figure 6B). Specifically, enCj4Cas9 and ultraCj4Cas9 exhibited 2.03-fold and 2.14-fold higher overall activity, respectively, relative to the wild-type (Figure 6C). Western blot analysis confirmed that the protein expression levels of all three variants were comparable, eliminating the possibility that increased activity was due to differential expression (Figure 6D). These results highlight the superior performance of the engineered Cj4Cas9 variants in genome editing applications.\u003c/p\u003e\n\u003cp\u003eWe focused on enCj4Cas9 in subsequent studies. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we compared the editing activity of WT Cj4Cas9, enCj4Cas9, and SpRY, a previously engineered SpCas9 variant with relaxed PAM specificity \u003csup\u003e28\u003c/sup\u003e. We designed a panel of 13 endogenous sites containing NNNGRY PAMs. Five days post-transfection, genomic DNA was extracted and subjected to targeted deep sequencing. The results revealed that Cj4Cas9 exhibited editing activity comparable to that of SpRY across the tested sites (Figure 7A-B). To ensure consistency, all three Cas9 orthologs were cloned into identical construct backbones, and Western blot analysis confirmed comparable protein expression levels among Cj4Cas9, enCj4Cas9, and SpRY (Figure 7C).\u003c/p\u003e\n\u003cp\u003eThe PAM preference of the enCj4Cas9 variant was further characterized using the GFP-activation assay. Intriguingly, the results demonstrated that enCj4Cas9 preferentially recognized a simple NNNGG PAM (Figure 7D-E). Additionally, we investigated the specificity of enCj4Cas9 by designing sgRNAs with dinucleotide mismatches targeting the AAVS1-TS1 locus. The results indicated that Cj4Cas9 and enCj4Cas9 exhibited similar specificity profiles (Figure S7A, lower panel). To assess genome-wide off-target effects, we performed the GUIDE-seq assay using a sgRNA targeting the EMX1-TS10 locus. Five days after co-transfecting HEK293T cells with Cj4Cas9/sgRNA-expressing plasmids and GUIDE-seq oligos, genomic DNA was extracted and prepared for deep sequencing. We identified an off-target site identical to the Cj4Cas9 off-target site (Figure S7B). In summary, enCj4Cas9 demonstrated specificity comparable to that of Cj4Cas9.\u003c/p\u003e\n\u003cp\u003e2.9 Enhanced Cj4Cas9 activity by fusion to non-specific DNA-binding proteins\u003c/p\u003e\n\u003cp\u003eIn parallel, to explore additional strategies for enhancing activity, we constructed fusion proteins by linking non-sequence-specific DNA-binding domains (DBDs), Sso7d and HMG-D, to Cj4Cas9 (Figure S11A), as previously described by Li et al. \u003csup\u003e23\u003c/sup\u003e. Although these DBDs improved editing activity at certain loci, neither fusion variant showed a significant overall enhancement in editing efficiency (Figure S11B-C). Western blot analysis confirmed comparable protein expression levels among the three variants, ruling out expression differences as the cause of increased activity (Figure S11D). These findings suggest that the intrinsic structural modifications in enCj4Cas9 and ultraCj4Cas9 play a more pivotal role in enhancing activity compared to fusion strategies involving DBDs.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAmong the numerous CRISPR-Cas9 tools developed to date, CjCas9 stands out due to its exceptionally compact size (984 amino acids), which is smaller than other Cas9 orthologs. To expand its targeting scope, we and others have previously identified five closely related CjCas9 orthologs that exhibit activity in human cells. For instance, Cj2Cas9 and Cj3Cas9 recognize an N4CYA PAM \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, while Hsp1Cas9, Hsp2Cas9, and CcuCas9 recognize N4RAA, N4CC, and N4CNA PAMs, respectively \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In this study, we screened a panel of 11 CjCas9 orthologs and identified seven active nucleases, which displayed highly diverse PAM preferences, consistent with our earlier findings \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. These orthologs represent a valuable resource for future genetic engineering efforts aimed at developing novel genome-editing tools with expanded capabilities.\u003c/p\u003e \u003cp\u003eAmong these orthologs, Cj4Cas9 is particularly noteworthy due to its compact size (985 amino acids) and unique PAM preference (NNNGRY). Its small genome size makes it highly suitable for AAV delivery, a critical advantage for therapeutic applications. Moreover, Cj4Cas9 demonstrated robust editing efficiency in both in vitro and in vivo settings, including the successful disruption of the \u003cem\u003eTyr\u003c/em\u003e gene in mouse zygotes and the \u003cem\u003ePcsk9\u003c/em\u003e gene in adult mice. To further enhance its utility, we engineered a high-activity variant, enCj4Cas9, by introducing L58K/D900K mutations, which resulted in a two-fold increase in nuclease activity compared to the wild-type enzyme. Notably, enCj4Cas9 exhibited a simplified PAM preference (NNNGG), significantly broadening its targeting scope. These advancements highlight the potential of Cj4Cas9 and its engineered variants for more efficient genome-editing applications in both research and therapy.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we developed a novel small Cj4Cas9 that can efficiently induce genome editing in vivo. We further generated enCj4Cas9 with improved activity and a larger targeting scope. Cj4Cas9 and enCj4Cas9 are promising tools for basic research and therapeutic applications.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e3.1 Animals\u003c/p\u003e\n\u003cp\u003eAge-matched C57BL/6 and ICR mice (Institute of Laboratory Animal Sciences, JSJ, China) were used as controls. All experimental mice were maintained in the animal facility of School of Life Sciences, Fudan University. Mice were housed in a 12-h light/dark cycle, with enough water and food. The mouse procedures were approved by the Institutional Animal Care and Use Committee of Fudan University (No.: 2024JS075). All procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.\u003c/p\u003e\n\u003cp\u003e3.2 Cas9 expression plasmid construction\u003c/p\u003e\n\u003cp\u003eThe plasmid Cas9-AAV was amplified by the primers Cas9-F/Cas9-R to obtain the Cas9-AAV backbone. The human codon optimized Cas9 gene (Supplementary file 1) was synthesized by HuaGene (Shanghai, China) and cloned into the Cas9-AAV backbone by the NEBuilder assembly tool (NEB) according to the manufacturer\u0026rsquo;s instructions. Sequences of Cas9 were confirmed by Sanger sequencing (Azenta, Suzhou, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.3 sgRNA expression plasmid construction\u003c/p\u003e\n\u003cp\u003eThe sgRNA expression plasmids were constructed by ligating sgRNA into the Bbs I-digested U6-Cj_scaffold plasmid，which is the same as CjCas9-scaffold. The primer sequences and target sequences are listed in Supplementary file 2 and Supplementary file 3, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.4 Cell culture and transfection\u003c/p\u003e\n\u003cp\u003eThe cell culture reagents were purchased from Gibco unless otherwise indicated. HEK293T and N2a cell lines were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM). All cell cultures were supplemented with 10% fetal bovine serum (FBS) (Gibco) that was inactivated at 56 \u0026deg;C for 30 min and 1% penicillin-streptomycin (Gibco). All cells were cultured in a humidified incubator at 37 \u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. All cell line identities were validated by STR profiling (ATCC) and repeatedly tested for mycoplasma by PCR.\u003c/p\u003e\n\u003cp\u003eHEK293T and N2a cells were transfected with Lipofectamine 2000 (Life Technologies) according to the manufacturer\u0026rsquo;s instructions. For transient transfection, a total of 500 ng Cas9-expressing plasmid and 300 ng sgRNA plasmid were co-transfected into a 24-well plate. For Cas9 PAM sequence screening, 1.2\u0026times;10\u003csup\u003e7\u003c/sup\u003e HEK293T cells were transfected with 10 \u0026mu;g of Cas9 plasmid and 5 \u0026mu;g of sgRNA plasmid in 10 cm dishes.\u003c/p\u003e\n\u003cp\u003e3.5 PAM discovery assay\u003c/p\u003e\n\u003cp\u003eThe PAM discovery assays were performed essentially as previously described, using a library of 7N. Transfected library cells with a certain percentage of GFP-positive cells were collected by centrifugation at 1000 rpm for 5 min and resuspended in PBS. Then, GFP-positive cells were collected by flow cytometry and cultured in six-well plates. Five days after culture, we extracted the genome and built deep sequencing library.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.6 Genome editing and deep sequencing analysis of indels for endogenous sites\u003c/p\u003e\n\u003cp\u003eCells were seeded into 24-well plates one day prior to transfection and transfected at 70\u0026ndash;80% confluency using Lipofectamine 2000 (Life Technologies) following the manufacturer\u0026rsquo;s recommended protocol. For genome editing, 10\u003csup\u003e6\u003c/sup\u003e cells were transfected with a total of 500 ng of Cas9 plasmid and 300 ng of sgRNA plasmid in 28-well plates. Six days after transfection, the cells were harvested, and genomic DNA was extracted in QuickExtract DNA Extraction Solution (Epicenter). To measure indel frequencies, the target sites were amplified by two rounds of nested PCR to add the Illumina adaptor sequence. The PCR products (200-300 bp in length) were gel-extracted by a Gel Extraction Kit (TIANGEN) for deep sequencing.\u003c/p\u003e\n\u003cp\u003e3.7 mRNA and gRNA preparation\u003c/p\u003e\n\u003cp\u003eThe Cj4Cas9 and GFP were PCR amplified with primers containing T7 promoter and 75 nt poly-A tail with KOD FX (TOYOBO) and transcribed \u003cem\u003ein vitro\u003c/em\u003e using the RNA transcription kit (novoprotein). Cj4Cas9 gRNA was PCR amplified with primers containing T7 promoter. These \u003cem\u003ein vitro\u003c/em\u003e transcribed mRNA and gRNA were stored at -80\u0026deg;C after purification with Monarch RNA cleanup kit (NEB) according to the manufacture\u0026rsquo;s protocol. Aliquoting was necessary for convenience and longer storage.\u003c/p\u003e\n\u003cp\u003e3.8 Microinjection of mouse zygotes and genotyping\u003c/p\u003e\n\u003cp\u003eGroups of 30 pronuclear-stage zygotes were injected with Cj4Cas9 mRNA (25 ng/\u0026micro;L) and sgRNA (12.5 ng/\u0026micro;L). The zygotes were cultured in KSOM at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The zygotes were collected individually with QuickExtract DNA Extraction Solution (Epicenter) when developed into blastocyst stage to analyze gene editing efficiencies. Embryo lysis were subjected to PCR amplification. Amplicons containing target sequences were analyzed by deep sequencing.\u003c/p\u003e\n\u003cp\u003e3.9 GUIDE-seq Off-target assay\u003c/p\u003e\n\u003cp\u003eWe performed a GUIDE-seq experiment with some modifications to the original protocol, as described\u003csup\u003e19\u003c/sup\u003e. On the day of the experiment, 2x10\u003csup\u003e5\u003c/sup\u003e HEK293T cells per target site were harvested and washed in PBS and transfected with 500 ng of Cas9 plasmid, 500 ng of sgRNA plasmid and 100 pmol annealed GUIDE-seq oligonucleotides through the Neon Transfection System. The electroporation voltage, width, and number of pulses were 1150 V, 20 ms, and 2 pulses, respectively. Genomic DNA was extracted with the DNeasy Blood and Tissue kit (QIAGEN) for 6-10 days according to cell proliferation after electroporation according to the manufacturer\u0026rsquo;s protocol. The genome library was prepared and subjected to deep sequencing.\u003c/p\u003e\n\u003cp\u003e3.10 Histology and Serum Analysis\u003c/p\u003e\n\u003cp\u003eTissues were fixed using 4% PFA at 4 \u0026deg;C overnight and dehydrated the next day before paraffinization. Paraffin blocks were cut into 5 \u0026micro;m thick sections, deparaffinized with xylene, and rehydrated. Sections were stained for DAPI and examined for transduction efficacy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSerum levels of LDL-C were evaluated by surfactant removal method (Gcell) following the manufacturer\u0026apos;s instructions. Similarly, the Total Cholesterol Assay Kit (Gcell) was utilized to measure CHO levels in the serum.\u003c/p\u003e\n\u003cp\u003e3.11 Statistics\u003c/p\u003e\n\u003cp\u003eAll statistics used in this study were performed on at least n = 3 biologically independent experiments and calculated using an unpaired or paired two-tailed Student\u0026apos;s t-test using R package dplyr. Detailed information on samples and experimental replicates can be found in the figure legends. p values less than 0.05 were considered significant, denoted as \u0026lowast;p \u0026lt; 0.05, \u0026lowast;\u0026lowast;p \u0026lt; 0.01, and \u0026lowast;\u0026lowast;\u0026lowast;p \u0026lt; 0.001.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e TY. Wang conceived and designed the experiments. TY. Wang, YF. Tian, Y. Zhang, MR. Li, J. Luo, and BW. Chen performed the experiments. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by grants from the National Key Research and Development Program of China (2023YFC2705600, 2023YFC2705602, 2021YFA0910602); the National Natural Science Foundation of China (82370254, 82070258); and the Science and Technology Research Program of Shanghai (24HC2810100, 23ZR1426000).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eInformed consents were obtained from all subjects involved in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eJinek, M.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e337\u003c/strong\u003e, 816-821 (2012). https://doi.org:10.1126/science.1225829\u003c/li\u003e\n \u003cli\u003eRan, F. 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P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e368\u003c/strong\u003e, 290-296 (2020). https://doi.org:10.1126/science.aba8853\u003c/li\u003e\n \u003cli\u003eWei, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Closely related type II-C Cas9 orthologs recognize diverse PAMs. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e (2022). https://doi.org:10.7554/eLife.77825\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":"CRISPR, Type II-C Cas9, AAV, PCSK9, Tyr","lastPublishedDoi":"10.21203/rs.3.rs-6451526/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6451526/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNatural CRISPR-Cas9 systems provide a rich resource for developing genome editing tools with diverse properties, including genome size, protospacer preference, and PAM specificity. In this study, we screened a panel of 11 Cas9 nucleases orthologous to CjCas9 using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 emerges as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5\u0026rsquo;-NNNGRY-3\u0026rsquo;). Cj4Cas9 demonstrates efficient disruption of the \u003cem\u003eTyr\u003c/em\u003e gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. To enhance its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings highlight the potential of Cj4Cas9 and its high-activity variants for both fundamental research and therapeutic applications.\u003c/p\u003e","manuscriptTitle":"In vivo genome editing with a novel Cj4Cas9","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 08:36:19","doi":"10.21203/rs.3.rs-6451526/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":"2a3ce37a-4147-4647-8691-df7195fd9219","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48241473,"name":"Biological sciences/Molecular biology/CRISPR-Cas systems/CRISPR-Cas9 genome editing"},{"id":48241474,"name":"Biological sciences/Biotechnology"}],"tags":[],"updatedAt":"2026-02-04T08:20:17+00:00","versionOfRecord":{"articleIdentity":"rs-6451526","link":"https://doi.org/10.1038/s42003-025-09430-9","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2025-12-30 05:00:00","publishedOnDateReadable":"December 30th, 2025"},"versionCreatedAt":"2025-05-13 08:36:19","video":"","vorDoi":"10.1038/s42003-025-09430-9","vorDoiUrl":"https://doi.org/10.1038/s42003-025-09430-9","workflowStages":[]},"version":"v1","identity":"rs-6451526","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6451526","identity":"rs-6451526","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}