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.
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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.
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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).
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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.
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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
