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Compact RNA editors with small Cas13 proteins

Nature Biotechnology volume 40, pages 194–197 (2022)Cite this article

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Abstract

CRISPR–Cas13 systems have been developed for precise RNA editing, and can potentially be used therapeutically when temporary changes are desirable or when DNA editing is challenging. We have identified and characterized an ultrasmall family of Cas13b proteins—Cas13bt—that can mediate mammalian transcript knockdown. We have engineered compact variants of REPAIR and RESCUE RNA editors by functionalizing Cas13bt with adenosine and cytosine deaminase domains, and demonstrated packaging of the editors within a single adeno-associated virus.

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Fig. 1: Cas13bt is a functional family of small Cas nucleases.
Fig. 2: RNA editing with Cas13bt.

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Data availability

Deep sequencing data from whole-transcriptome sequencing were deposited as a BioProject under Project ID PRJNA641934. Data from the main figures are available in the Supplementary Information.

Code availability

All Python scripts used for data analysis are available in a GitHub repository found at https://github.com/fengzhanglab/Cas13bt-analysis.

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Acknowledgements

We thank E. Eloe-Fadrosh for generously sharing JGI data from study Ga0114919; D. L. Valentine and J. Tarn for generously sharing JGI data from study Ga0180434; E. Puccio for assistance with AAV production; F. E. Demircioglu for assistance with protein purification; members of the laboratory of F.Z. for advice and discussions, R. Macrae for discussion and editing of the manuscript, and R. Belliveau for technical support. S.K. is supported by a National Science Foundation Graduate Research Fellowship. F.Z. is supported by NIH grants (1R01-HG009761, 1R01-MH110049 and 1DP1-HL141201), HHMI, Open Philanthropy, The G. Harold and Leila Y. Mathers Charitable, Bill and Melinda Gates, and Edward Mallinckrodt, Jr Foundations, the Poitras Center for Psychiatric Disorders Research at MIT, the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, the Yang-Tan Center for Molecular Therapeutics, and by L. Yang, the Phillips family and J. and P. Poitras.

Author information

Author notes

  1. These authors contributed equally: Soumya Kannan, Han Altae-Tran.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Cambridge, MA, USA

    Soumya Kannan, Han Altae-Tran, Xin Jin, Victoria J. Madigan, Rachel Oshiro & Feng Zhang

  2. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Soumya Kannan, Han Altae-Tran, Xin Jin, Victoria J. Madigan, Rachel Oshiro & Feng Zhang

  3. McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA

    Soumya Kannan, Han Altae-Tran, Xin Jin, Victoria J. Madigan, Rachel Oshiro & Feng Zhang

  4. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Soumya Kannan, Han Altae-Tran, Xin Jin, Victoria J. Madigan, Rachel Oshiro & Feng Zhang

  5. Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA, USA

    Soumya Kannan, Han Altae-Tran, Xin Jin, Victoria J. Madigan, Rachel Oshiro & Feng Zhang

  6. Society of Fellows, Harvard University, Cambridge, MA, USA

    Xin Jin

  7. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA

    Xin Jin

  8. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA

    Kira S. Makarova & Eugene V. Koonin

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Contributions

S.K., H.A.-T. and F.Z. conceived the project and designed the computational pipeline and experiments. H.A.-T. performed computational searching and phylogenetic analysis with input from K.S.M. and E.V.K. S.K. and H.A.-T. performed the essential gene screen experiment and H.A.-T. analyzed the results. X.J. performed the transcriptome-wide specificity sequencing experiment, and S.K. analyzed the results. V.M. produced AAV and assisted with design of AAV constructs and experiments. S.K. performed and analyzed results from all other experiments with assistance from X.J., R.O. and V.M. S.K., H.A.-T. and F.Z. drafted the manuscript with input from all authors.

Corresponding author

Correspondence to Feng Zhang.

Ethics declarations

Competing interests

F.Z. is a co-founder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies and Sherlock Biosciences. S.K., H.A.-T. and F.Z. are co-inventors on US provisional patent application 62/905,645 relating to the Cas proteins described in this manuscript. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Biotechnology thanks Magdy Mahfouz, Mitchell O’Connell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–10, Tables 1–11, Note 1 and References.

Reporting Summary

Supplementary Tables

Supplementary Tables 4–6, 8 and 11.

Supplementary Data

Unprocessed gel for Supplementary Fig. 4a.

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Kannan, S., Altae-Tran, H., Jin, X. et al. Compact RNA editors with small Cas13 proteins. Nat Biotechnol 40, 194–197 (2022). https://doi.org/10.1038/s41587-021-01030-2

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