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.

  • Brief Communication
  • Published:

Compact RNA editors with small Cas13 proteins

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

Subjects

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.

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

Similar content being viewed by others

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.

References

  1. Terns, M. P. CRISPR-based technologies: impact of RNA-targeting systems. Mol. Cell 72, 404–412 (2018).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  3. Abudayyeh, O. O. et al. A cytosine deaminase for programmable single-base RNA editing. Science 365, 382–386 (2019).

    Article  CAS  Google Scholar 

  4. Dong, J. Y., Fan, P. D. & Frizzel, R. A. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum. Gene Ther. 7, 2101–2112 (1996).

    Article  CAS  Google Scholar 

  5. Wu, Z., Yang, H. & Colosi, P. Effect of genome size on AAV vector packaging. Mol. Ther. 18, 80–86 (2010).

    Article  CAS  Google Scholar 

  6. 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 (2017).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  8. 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 

  9. Matthews, M. M. et al. Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat. Struct. Mol. Biol. 23, 426–433 (2016).

    Article  CAS  Google Scholar 

  10. MacDonald, B. T., Tamai, K. & He, X. Wnt/β-catenin signaling: components, mechanisms, and diseases. Dev. Cell 17, 9–26 (2009).

    Article  CAS  Google Scholar 

  11. Apte, U. et al. Beta-catenin activation promotes liver regeneration after acetaminophen-induced injury. Am. J. Pathol. 175, 1056–1065 (2009).

    Article  CAS  Google Scholar 

  12. Bhushan, B. et al. Pro-regenerative signaling after acetaminophen-induced acute liver injury in mice identified using a novel incremental dose model. Am. J. Pathol. 184, 3013–3025 (2014).

    Article  CAS  Google Scholar 

  13. Kattenhorn, L. M. et al. Adeno-associated virus gene therapy for liver disease. Hum. Gene Ther. 27, 947–961 (2016).

    Article  CAS  Google Scholar 

  14. Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172–1179 (2017).

    Article  CAS  Google Scholar 

  15. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  Google Scholar 

  16. Keegan, K. P., Glass, E. M. & Meyer, F. MG-RAST, a metagenomics service for analysis of microbial community structure and function. Methods Mol. Biol. 1399, 207–233 (2016).

    Article  CAS  Google Scholar 

  17. Nordberg, H. et al. The genome portal of the Department of Energy Joint Genome Institute: 2014 updates. Nucleic Acids Res. 42, 26–31 (2014).

    Article  Google Scholar 

  18. Chen, I. M. A. et al. The IMG/M data management and analysis system v.6.0: new tools and advanced capabilities. Nucleic Acids Res. 49, D751–D763 (2020).

    Article  Google Scholar 

  19. Shmakov, S. A., Makarova, K. S., Wolf, Y. I., Severinov, K. V. & Koonin, E. V. Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis. Proc. Natl Acad. Sci. USA 115, E5307–E5316 (2018).

    Article  CAS  Google Scholar 

  20. Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).

    Article  CAS  Google Scholar 

  21. Steinegger, M. & Söding, J. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat. Biotechnol. 35, 1026–1028 (2017).

    Article  CAS  Google Scholar 

  22. Steinegger, M. & Söding, J. Clustering huge protein sequence sets in linear time. Nat. Commun. 9, 2542 (2018).

    Article  Google Scholar 

  23. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    Article  CAS  Google Scholar 

  24. Steinegger, M. et al. HH-suite3 for fast remote homology detection and deep protein annotation. BMC Bioinformatics 20, 1–15 (2019).

    Article  CAS  Google Scholar 

  25. Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2019).

    Article  Google Scholar 

  26. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2 - approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).

    Article  Google Scholar 

  27. Joung, J. et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).

    Article  CAS  Google Scholar 

  28. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

    Article  CAS  Google Scholar 

  29. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).

    Article  CAS  Google Scholar 

  30. Hrvatin, S., Deng, F., O’Donnell, C. W., Gifford, D. K. & Melton, D. A. MARIS: method for analyzing RNA following intracellular sorting. PLoS ONE 9, e89459 (2014).

    Article  Google Scholar 

  31. Veeman, M. T., Slusarski, D. C., Kaykas, A., Louie, S. H. & Moon, R. T. Zebrafish Prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr. Biol. 13, 680–685 (2003).

    Article  CAS  Google Scholar 

  32. Glusman, G., Caballero, J., Mauldin, D. E., Hood, L. & Roach, J. C. Kaviar: an accessible system for testing SNV novelty. Bioinformatics 27, 3216–3217 (2011).

    Article  CAS  Google Scholar 

  33. Shen, S., Troupes, A. N., Pulicherla, N. & Asokan, A. Multiple roles for sialylated glycans in determining the cardiopulmonary tropism of adeno-associated Virus 4. J. Virol. 87, 13206–13213 (2013).

    Article  CAS  Google Scholar 

  34. Grieger, J. C., Choi, V. W. & Samulski, R. J. Production and characterization of adeno-associated viral vectors. Nat. Protoc. 1, 1412–1428 (2006).

    Article  CAS  Google Scholar 

Download references

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

Authors
  1. Soumya Kannan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Han Altae-Tran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xin Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Victoria J. Madigan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rachel Oshiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kira S. Makarova
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eugene V. Koonin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Feng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41587-021-01030-2

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