Abstract
CRISPR–Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.
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Data availability
Publicly available datasets used in this study were as follows: GRCh38.p5. RNA-seq data are available under GEO accession number GSE168246. Source data are provided with this paper. Any other data can be obtained from the corresponding author upon reasonable request.
Code availability
Code that supports the findings of this study is available in the Supplementary Information.
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Acknowledgements
We thank M.-m. Poo for helpful discussions and insightful comments on this manuscript; H. Yang and B. Wang from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, for helpful discussions and technical assistance; L.-L. Chen from CAS Center for Excellence in Molecular Cell Science for the gift of the RanCas13b and dRanCas13b plasmids; and Y. Wang, Y. Zhang and Q. Hu from the Optical Imaging facility and S. Qian, H. Wu and L. Quan from the FACS facility of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences for technical support. We thank N. Zhong and L. Xie for technical assistance. This work was supported by HUIGENE Therapeutics Co., Ltd. (H.T.), Lingang Laboratory (LG202106-01-02) (H.Y.), Chinese National Science and Technology major project R&D Program of China (2018YFC2000101) (H.Y.), Strategic Priority Research Program of Chinese Academy of Science (XDB32060000) (H.Y.), National Natural Science Foundation of China (31871502, 31901047, 31925016, 91957122 and 82021001) (H.Y.), Basic Frontier Scientific Research Program of Chinese Academy of Sciences From 0 to 1 original innovation project (ZDBS-LY-SM001) (H.Y.), Shanghai Municipal Science and Technology Major Project (2018SHZDZX05) (H.Y.), Shanghai City Committee of Science and Technology Project (18411953700, 18JC1410100, 19XD1424400 and 19YF1455100) (H.Y.) and the International Partnership Program of Chinese Academy of Sciences (153D31KYSB20170059) (H.Y.).
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Competing interests
H.T. discloses a patent application (PCT/CN2021/121926) related to the Cas proteins described in this manuscript. H.T. is an employee of HuiGene Therapeutics Co., Ltd. H.Z. is now an employee of HuiEdit Therapeutics Co., Ltd. H.Y. is a founder of HuiGene Therapeutics Co., Ltd. and HuiEdit Therapeutics Co., Ltd. The remaining authors declare no competing interests.
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Tong, H., Huang, J., Xiao, Q. et al. High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects.
Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01419-7
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DOI: https://doi.org/10.1038/s41587-022-01419-7
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