Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Biotechnology volume 41, pages 108–119 (2023)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Evaluation of collateral effects in transiently transfected HEK293T cells using a dual-fluorescence reporter.
Fig. 2: Rational mutagenesis of Cas13d to eliminate collateral activity.
Fig. 3: Efficient and specific interference activity of hfCas13d in HEK293 cells.
Fig. 4: Transcriptome-wide collateral effect analysis for Cas13d and hfCas13d.
Fig. 5: Cellular and in vivo consequences of collateral cleavage and their elimination.
Fig. 6: Generation of hfCas13X by rational mutagenesis and its efficacy in vivo.

Similar content being viewed by others

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.

References

  1. Knott, G. J. & Doudna, J. A. CRISPR-Cas guides the future of genetic engineering. Science 361, 866–869 (2018).

    Article  CAS  Google Scholar 

  2. Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR-Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 490–507 (2019).

    Article  CAS  Google Scholar 

  3. Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016).

    Article  Google Scholar 

  4. Meeske, A. J., Nakandakari-Higa, S. & Marraffini, L. A. Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage. Nature 570, 241–245 (2019).

    Article  CAS  Google Scholar 

  5. Abudayyeh, O. O. et al. RNA targeting with CRISPR-Cas13. Nature 550, 280–284 (2017).

    Article  Google Scholar 

  6. Cox, D. B. T. et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017).

    Article  CAS  Google Scholar 

  7. Konermann, S. et al. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell 173, 665–676.e614 (2018).

    Article  CAS  Google Scholar 

  8. Yang, L. Z. et al. Dynamic imaging of RNA in living cells by CRISPR-Cas13 systems. Mol. Cell 76, 981–997.e987 (2019).

    Article  CAS  Google Scholar 

  9. Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).

    Article  CAS  Google Scholar 

  10. Gootenberg, J. S. et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360, 439–444 (2018).

    Article  CAS  Google Scholar 

  11. Ackerman, C. M. et al. Massively multiplexed nucleic acid detection with Cas13. Nature 582, 277–282 (2020).

    Article  CAS  Google Scholar 

  12. Aman, R. et al. RNA virus interference via CRISPR/Cas13a system in plants. Genome Biol. 19, 1 (2018).

    Article  Google Scholar 

  13. Jing, X. et al. Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing. Nucleic Acids Res. 46, e90 (2018).

    Article  Google Scholar 

  14. Kushawah, G. et al. CRISPR-Cas13d induces efficient mRNA knockdown in animal embryos. Dev. Cell 54, 805–817.e807 (2020).

    Article  CAS  Google Scholar 

  15. Huynh, N., Depner, N., Larson, R. & King-Jones, K. A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila. Genome Biol. 21, 279 (2020).

    Article  CAS  Google Scholar 

  16. Abbott, T. R. et al. Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and influenza. Cell 181, 865–876.e812 (2020).

    Article  CAS  Google Scholar 

  17. Li, S. et al. Screening for functional circular RNAs using the CRISPR-Cas13 system. Nat. Methods 18, 51–59 (2021).

    Article  Google Scholar 

  18. Blanchard, E. L. et al. Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents. Nat. Biotechnol. 39, 717–726 (2021).

    Article  CAS  Google Scholar 

  19. Ozcan, A. et al. Programmable RNA targeting with the single-protein CRISPR effector Cas7-11. Nature 597, 720–725 (2021).

    Article  CAS  Google Scholar 

  20. Xu, C. et al. Programmable RNA editing with compact CRISPR-Cas13 systems from uncultivated microbes. Nat. Methods 18, 499–506 (2021).

    Article  CAS  Google Scholar 

  21. He, B. et al. Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver. Protein Cell 11, 518–524 (2020).

    Article  CAS  Google Scholar 

  22. Zhou, C. et al. CasRx-mediated RNA targeting prevents choroidal neovascularization in a mouse model of age-related macular degeneration. Natl Sci. Rev. 7, 835–837 (2020).

    Article  CAS  Google Scholar 

  23. Zhou, H. et al. Glia-to-neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice. Cell 181, 590–603.e516 (2020).

    Article  CAS  Google Scholar 

  24. Powell, J. E. et al. Targeted gene silencing in the nervous system with CRISPR-Cas13. Sci. Adv. 8, eabk2485 (2022).

    Article  CAS  Google Scholar 

  25. Porto, E. M., Komor, A. C., Slaymaker, I. M. & Yeo, G. W. Base editing: advances and therapeutic opportunities. Nat. Rev. Drug Discov. 19, 839–859 (2020).

    Article  CAS  Google Scholar 

  26. East-Seletsky, A. et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 538, 270–273 (2016).

    Article  CAS  Google Scholar 

  27. Varble, A. & Marraffini, L. A. Three new Cs for CRISPR: collateral, communicate, cooperate. Trends Genet. 35, 446–456 (2019).

    Article  CAS  Google Scholar 

  28. Buchman, A. B. et al. Programmable RNA targeting using CasRx in flies. CRISPR J. 3, 164–176 (2020).

    Article  CAS  Google Scholar 

  29. Wang, Q. et al. The CRISPR-Cas13a gene-editing system induces collateral cleavage of RNA in glioma cells. Adv. Sci. 6, 1901299 (2019).

    Article  CAS  Google Scholar 

  30. Wang, L., Zhou, J., Wang, Q., Wang, Y. & Kang, C. Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein. Theranostics 11, 649–664 (2021).

    Article  CAS  Google Scholar 

  31. Ai, Y., Liang, D. & Wilusz, J. E. CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells. Nucleic Acids Res. 50, e65 (2022).

    Article  CAS  Google Scholar 

  32. Kelley, C.P., Haerle, M.C. & Wang, E.T. Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells. Preprint at bioRxiv https://doi.org/10.1101/2021.12.20.473384 (2021).

  33. Shi, P. et al. RNA-guided cell targeting with CRISPR/RfxCas13d collateral activity in human cells. Preprint at bioRxiv https://doi.org/10.1101/2021.11.30.470032 (2021).

  34. Li, Y. et al. Collateral cleavage of 28s rRNA by RfxCas13d causes death of mice. Preprint at bioRxiv https://doi.org/10.1101/2022.01.17.476700 (2022).

  35. Zhang, C. et al. Structural basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d. Cell 175, 212–223.e217 (2018).

    Article  CAS  Google Scholar 

  36. Liu, L. et al. Two distant catalytic sites are responsible for C2c2 RNase activities. Cell 168, 121–134.e112 (2017).

    Article  CAS  Google Scholar 

  37. Zhang, B. et al. Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d. Nat. Commun. 10, 2544 (2019).

    Article  Google Scholar 

  38. Slaymaker, I. M. et al. High-resolution structure of Cas13b and biochemical characterization of RNA targeting and cleavage. Cell Rep. 26, 3741–3751.e3745 (2019).

    Article  CAS  Google Scholar 

  39. Liu, L. et al. The molecular architecture for RNA-guided RNA cleavage by Cas13a. Cell 170, 714–726.e710 (2017).

    Article  CAS  Google Scholar 

  40. Zhang, Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9, 40 (2008).

    Article  Google Scholar 

  41. Kiga, K. et al. Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria. Nat. Commun. 11, 2934 (2020).

    Article  CAS  Google Scholar 

  42. Steinberg, D. & Witztum, J. L. Inhibition of PCSK9: a powerful weapon for achieving ideal LDL cholesterol levels. Proc. Natl Acad. Sci. USA 106, 9546–9547 (2009).

    Article  CAS  Google Scholar 

  43. Rossidis, A. C. et al. In utero CRISPR-mediated therapeutic editing of metabolic genes. Nat. Med. 24, 1513–1518 (2018).

    Article  CAS  Google Scholar 

  44. Chan, J. C. et al. A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates. Proc. Natl Acad. Sci. USA 106, 9820–9825 (2009).

    Article  CAS  Google Scholar 

  45. Shen, X., Storm, T. & Kay, M. A. Characterization of the relationship of AAV capsid domain swapping to liver transduction efficiency. Mol. Ther. 15, 1955–1962 (2007).

    Article  CAS  Google Scholar 

  46. Smargon, A. A. et al. Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28. Mol. Cell 65, 618–630.e617 (2017).

    Article  CAS  Google Scholar 

  47. Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).

    Article  Google Scholar 

  48. Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    Article  Google Scholar 

  49. Huang da, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  Google Scholar 

  50. Huang da, W., Sherman, B. T. & Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

    Article  Google Scholar 

Download references

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

Authors
  1. Huawei Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jia Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qingquan Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bingbing He
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xue Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yuanhua Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiali Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dingyi Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zikang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Xuchen Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Wenqin Ying
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Runze Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yu Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Chunlong Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Yingsi Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Yanfei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Minqing Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Qifang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Mingxing Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Guoling Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Kailun Fang
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Hainan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Hui Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Peer review

Peer review information

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

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs 1–21, Tables 1 and 3–6 and Note 1.

Reporting Summary

Supplementary Table 2

Predicted gRNA-dependent off-target genes.

Supplementary Data 1

Source data for supplementary figures.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 2

Unprocessed gel.

Source Data Fig. 6

Unprocessed gel.

Unprocessed Gels Supplementary Figures

Unprocessed gels.

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tong, H., Huang, J., Xiao, Q. et al. High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects. Nat Biotechnol 41, 108–119 (2023). https://doi.org/10.1038/s41587-022-01419-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-022-01419-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research