In vivo genome editing with a novel Cj4Cas9

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In vivo genome editing with a novel Cj4Cas9

Communications Biology , Article number:  (2025) Cite this article

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Abstract

Natural CRISPR-Cas9 systems provides diverse properties for genome editing, yet finding compact variants remains a priority. In this study, we screened a panel of 11 CjCas9 orthologous using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 stood out 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. Seeking to further expand 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 establish Cj4Cas9 and its engineered variants for fundamental research and therapeutic applications.

Data availability

All NGS data have been uploaded to the NCBI Sequence Read Archive database under accession code PRJNA1307308. There are no restrictions on data availability. All raw data in this study were listed in Supplementary Data 4. Uncropped and unedited western blot images are provided in Supplementary Figs. S12–14.

References

  1. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Google Scholar 

  2. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Google Scholar 

  3. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Google Scholar 

  4. Wang, D. et al. Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning. Nat. Commun. 10, 4284 (2019).

    Google Scholar 

  5. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Google Scholar 

  6. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Google Scholar 

  7. Kan, Y., Ruis, B., Takasugi, T. & Hendrickson, E. A. Mechanisms of precise genome editing using oligonucleotide donors. Genome Res. 27, 1099–1111 (2017).

    Google Scholar 

  8. Hu, Z. et al. A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope. PLoS Biol. 18, e3000686 (2020).

    Google Scholar 

  9. Hu, Z. et al. Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity. Nucleic Acids Res. 49, 4008–4019 (2021).

    Google Scholar 

  10. Wang, S. et al. Compact SchCas9 recognizes the simple NNGR PAM. Adv. Sci. (Weinh.) 9, e2104789 (2022).

    Google Scholar 

  11. Edraki, A. et al. A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing. Mol. Cell 73, 714–726.e714 (2019).

    Google Scholar 

  12. Gasiunas, G. et al. A catalogue of biochemically diverse CRISPR-Cas9 orthologs. Nat. Commun. 11, 5512 (2020).

    Google Scholar 

  13. Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017).

    Google Scholar 

  14. Nakagawa, R. et al. Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range. Commun. Biol. 5, 211 (2022).

    Google Scholar 

  15. Ruta, G. V. et al. Eukaryotic-driven directed evolution of Cas9 nucleases. Genome Biol. 25, 79 (2024).

    Google Scholar 

  16. Chen, S. et al. Compact Cje3Cas9 for efficient in vivo genome editing and adenine base editing. CRISPR J. 5, 472–486 (2022).

    Google Scholar 

  17. Gao, S. et al. Genome editing with natural and engineered CjCas9 orthologs. Mol. Ther. 31, 1177–1187 (2023).

    Google Scholar 

  18. 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).

    Google Scholar 

  19. 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).

    Google Scholar 

  20. Wang, M. et al. Hypercompact TnpB and truncated TnpB systems enable efficient genome editing in vitro and in vivo. Cell Discov. 10, 31 (2024).

    Google Scholar 

  21. Musunuru, K. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429–434 (2021).

    Google Scholar 

  22. Sabatine, M. S. et al. Evolocumab and clinical outcomes in patients with cardiovascular disease. N. Engl. J. Med. 376, 1713–1722 (2017).

    Google Scholar 

  23. Yin, S. et al. Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies. Mol. Ther. 31, 744–759 (2023).

    Google Scholar 

  24. Jang, D. E. et al. Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency. Exp. Mol. Med. 50, 1–9 (2018).

    Google Scholar 

  25. 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).

    Google Scholar 

  26. 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).

    Google Scholar 

  27. Schmidheini, L. et al. Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility. Nat. Chem. Biol. 20, 333–343 (2024).

    Google Scholar 

  28. 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).

    Google Scholar 

  29. Wei, J. et al. Closely related type II-C Cas9 orthologs recognize diverse PAMs. Elife 11, https://doi.org/10.7554/eLife.77825 (2022).

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Acknowledgements

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).

Author information

Author notes

  1. These authors contributed equally: Tianyi Wang, Yafei Tian.

Authors and Affiliations

  1. Center for Medical Research and Innovation, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, China

    Tianyi Wang & Yongming Wang

  2. State Key Laboratory of Genetics and Development of Complex Phenotypes and MOE Engineering Research Center of Gene Technology, School of Life Sciences and Institute of Medical Genetics and Genomics, Fudan University, Shanghai, China

    Tianyi Wang, Yafei Tian, Rui Yin, Mengru Li, Jie Luo, Hongyan Chen & Daru Lu

  3. State Key Laboratory of Oncogenes and Related Genes, Center for Single-Cell Omics, School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yuan Yang & Chengdong Zhang

Authors

  1. Tianyi Wang
  2. Yafei Tian
  3. Rui Yin
  4. Mengru Li
  5. Jie Luo
  6. Yuan Yang
  7. Chengdong Zhang
  8. Hongyan Chen
  9. Yongming Wang
  10. Daru Lu

Contributions

T.Y. Wang conceived and designed the experiments. T.Y. Wang, Y.F. Tian, R. Yin, M.R. Li, J. Luo, and Y. Yang performed the experiments. Chendong Zhang, Hongyan Chen provided resources. Yongming Wang, and Daru Lu provided experimental guidance and supervision; Yongming Wang contributed to writing and reviewing the manuscript.

Corresponding authors

Correspondence to Chengdong Zhang, Hongyan Chen, Yongming Wang or Daru Lu.

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Competing interests

The authors declare no competing interests.

Informed consent

Informed consents were obtained from all subjects involved in the study.

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Peer review information

Communications Biology thanks Tristan Henser-Brownhill, Cem Kuscu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Yuhong Cao and Mengtan Xing. A peer review file is available.

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Wang, T., Tian, Y., Yin, R. et al. In vivo genome editing with a novel Cj4Cas9. Commun Biol (2025). https://doi.org/10.1038/s42003-025-09430-9

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  • DOI: https://doi.org/10.1038/s42003-025-09430-9

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