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

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BEING VIEWED BY OTHERS GENE EDITING WITH CRISPR-CAS12A GUIDES POSSESSING RIBOSE-MODIFIED PSEUDOKNOT HANDLES Article Open access 15 November 2021 MICAS9 INCREASES LARGE SIZE GENE KNOCK-IN

RATES AND REDUCES UNDESIRABLE ON-TARGET AND OFF-TARGET INDEL EDITS Article Open access 27 November 2020 PAM-FLEXIBLE GENOME EDITING WITH AN ENGINEERED CHIMERIC CAS9 Article Open access 04

October 2023 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. CHANGE HISTORY * _ 01 DECEMBER 2023 A Correction to this

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data. _Nat. Biotechnol._ 34, 483 (2016). Article  PubMed  Google Scholar  Download references 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. AUTHOR INFORMATION Author notes * These authors contributed equally: Tong Wu, Chang Liu, Siyuan Zou. * Deceased: Bowei

Yang. AUTHORS AND AFFILIATIONS * Department of Chemistry, The University of Chicago, Chicago, IL, USA Tong Wu, Chang Liu, Siyuan Zou, Ruitu Lyu, Hao Yan & Weixin Tang * 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 * Department of Biochemistry and

Molecular Biology, The University of Chicago, Chicago, IL, USA Bowei Yang & Minglei Zhao Authors * Tong Wu View author publications You can also search for this author inPubMed Google

Scholar * Chang Liu View author publications You can also search for this author inPubMed Google Scholar * Siyuan Zou View author publications You can also search for this author inPubMed 

Google Scholar * Ruitu Lyu View author publications You can also search for this author inPubMed Google Scholar * Bowei Yang View author publications You can also search for this author

inPubMed Google Scholar * Hao Yan View author publications You can also search for this author inPubMed Google Scholar * Minglei Zhao View author publications You can also search for this

author inPubMed Google Scholar * Weixin Tang View author publications You can also search for this author inPubMed Google Scholar 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. ETHICS DECLARATIONS 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. PEER REVIEW PEER REVIEW INFORMATION _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. ADDITIONAL INFORMATION PUBLISHER’S NOTE

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Tables 1–6 and Supplementary Figs. 1–18. REPORTING SUMMARY SUPPLEMENTARY DATA 1 Sequences of DNA oligos used to amplify genomic loci for amplicon sequencing. RIGHTS AND PERMISSIONS Springer

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CITE THIS ARTICLE 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).

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