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An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity

Nature Chemical Biology volume 19, pages 1384–1393 (2023)Cite this article

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An Author Correction to this article was published on 01 December 2023

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Abstract

Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f–sgRNA–DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.

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Fig. 1: Engineering AsCas12f for increased genome-editing efficiency.
Fig. 2: Genome editing facilitated by engineered AsCas12f systems.
Fig. 3: Cryo-EM structure of the AsCas12f–sgRNA–DNA complex.
Fig. 4: Structure-guided engineering of the AsCas12f gRNA.
Fig. 5: Genome-wide specificity of WT and engineered AsCas12f.

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Data availability

Sequencing data are available at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under accession number GSE211600 and the Sequence Read Archive (SRA) under accession number PRJNA962057. Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB, https://www.ebi.ac.uk/emdb) under accession code EMD-27801. The atomic model has been deposited to the Protein Data Bank (PDB, https://www.rcsb.org) under accession code 8DZJ.

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Acknowledgements

We thank all members of the Tang laboratory for discussion. We thank the staff at the University of Chicago Advanced Electron Microscopy Facility (RRID: SCR_019198) for helping with cryo-EM data collection. We thank the Research Computing Center at the University of Chicago for providing the computing resources of the Beagle 3 HPC cluster funded by the National Institutes of Health (NIH) (S10OD028655). This work was supported by the NIH under grant number R35GM143052 to M.Z. W.T. is supported by the Searle Scholars Program (SSP-2021-113), the Cancer Research Foundation Young Investigator Program, the American Cancer Society (RSG-22-043-01-ET), and the David & Lucile Packard Foundation.

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Author notes

  1. These authors contributed equally: Tong Wu, Chang Liu, Siyuan Zou.

  2. Deceased: Bowei Yang.

Authors and Affiliations

  1. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Tong Wu, Chang Liu, Siyuan Zou, Ruitu Lyu, Hao Yan & Weixin Tang

  2. Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Tong Wu, Siyuan Zou, Ruitu Lyu, Bowei Yang, Hao Yan, Minglei Zhao & Weixin Tang

  3. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA

    Bowei Yang & Minglei Zhao

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Contributions

All authors designed experiments and interpreted the data. T.W., M.Z. and W.T. conceived the project. T.W. and S.Z. performed protein engineering, gRNA engineering, cellular gene-editing and in vitro DNA cleavage assays with help and suggestions from H.Y. C.L. and B.Y. purified proteins. C.L. collected cryo-EM data and performed data analysis. R.L. assisted with GUIDE-seq data analysis. T.W., C.L., M.Z. and W.T. wrote the paper with input from all authors.

Corresponding authors

Correspondence to Minglei Zhao or Weixin Tang.

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Competing interests

T.W., S.Z. and W.T. are inventors on a US provisional patent application on enAsCas12f. T.W. is a shareholder of AccuraDX. All other authors declare no competing interests.

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Nature Chemical Biology thanks Jun-Jie Liu, Hyongbum Henry Kim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Tables 1–6 and Supplementary Figs. 1–18.

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Supplementary Data 1

Sequences of DNA oligos used to amplify genomic loci for amplicon sequencing.

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Wu, T., Liu, C., Zou, S. et al. An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity. Nat Chem Biol 19, 1384–1393 (2023). https://doi.org/10.1038/s41589-023-01380-9

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