High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects

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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.).

Author information

Author notes

  1. These authors contributed equally: Huawei Tong, Jia Huang, Qingquan Xiao, Bingbing He, Xue Dong, Yuanhua Liu.

Authors and Affiliations

  1. HuiGene Therapeutics Co., Ltd., Shanghai, China

    Huawei Tong & Hui Yang

  2. Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China

    Jia Huang, Qingquan Xiao, Bingbing He, Xue Dong, Yuanhua Liu, Dingyi Han, Zikang Wang, Xuchen Wang, Wenqin Ying, Runze Zhang, Yu Wei, Chunlong Xu, Yingsi Zhou, Minqing Cai, Qifang Wang, Mingxing Xue, Guoling Li, Kailun Fang, Hainan Zhang & Hui Yang

  3. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China

    Jia Huang, Qingquan Xiao, Dingyi Han, Xuchen Wang, Runze Zhang & Yu Wei

  4. Key Laboratory of Stem Cell Engineering and Regenerative Medicine of Fujian Provincial Colleges and Universities, Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China

    Xiali Yang

  5. Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai, China

    Chunlong Xu & Hui Yang

  6. Zhoupu Hospital Affiliated to Shanghai Health Medical College and Shanghai Key Laboratory of MolecularImaging, Shanghai, China

    Yanfei Li

  7. HuiEdit Therapeutics Co., Ltd., Shanghai, China

    Hainan Zhang & Hui Yang

Contributions

H.T., J.H. and H.Y. jointly conceived the project. H.T., J.H., Q.X., B.H. and X.D. designed and conducted experiments. Y.L. performed bulk RNA-seq analysis. J.H. and X.Y. performed microinjection and counted the mice every day. Q.X., X.W., R.Z. and Y.W. performed qPCR assays and participated in FACS. X.D. and D.H. participated in protein purification and in vitro cleavage assays. W.Y performed mouse embryo transfer. Y.L., M.C., Q.W. and M.X. assisted with plasmid construction. Z.W., C.X., Y.Z., G.L. and K.F. assisted with cell experiments. H.Y. supervised the whole project. H.T., H.Z. and H.Y. wrote the manuscript.

Corresponding authors

Correspondence to
Jia Huang, Hainan Zhang or Hui Yang.

Ethics declarations

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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Nature Biotechnology thanks Jonathan Gootenberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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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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