Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity

Connor A Tsuchida; Shouyue Zhang; Mohammad Saffari Doost; Yuqian Zhao; Jia Wang; Elizabeth O'Brien; Huan Fang; Cheng-Ping Li; Danyuan Li; Zhuo-Yan Hai; Jonathan Chuck; Julian Brötzmann; Araz Vartoumian; David Burstein; Xiao-Wei Chen; Eva Nogales; Jennifer A Doudna; Jun-Jie Gogo Liu

doi:10.1016/j.molcel.2022.02.002

Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity

Mol Cell. 2022 Mar 17;82(6):1199-1209.e6. doi: 10.1016/j.molcel.2022.02.002. Epub 2022 Feb 25.

Authors

Connor A Tsuchida¹, Shouyue Zhang², Mohammad Saffari Doost³, Yuqian Zhao², Jia Wang², Elizabeth O'Brien³, Huan Fang⁴, Cheng-Ping Li², Danyuan Li², Zhuo-Yan Hai², Jonathan Chuck⁵, Julian Brötzmann⁶, Araz Vartoumian³, David Burstein⁷, Xiao-Wei Chen⁴, Eva Nogales⁸, Jennifer A Doudna⁹, Jun-Jie Gogo Liu¹⁰

Affiliations

¹ University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, University of California, Berkeley, California 94720, USA; Innovative Genomics Institute, University of California, Berkeley, California 94720, USA.
² Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China; Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
³ Innovative Genomics Institute, University of California, Berkeley, California 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720, USA.
⁴ Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing 100871, China.
⁵ Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, Florida 33458, USA.
⁶ Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
⁷ School of Molecular Cell Biology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 6997801, Israel.
⁸ California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA; Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA.
⁹ University of California, Berkeley-University of California, San Francisco Graduate Program in Bioengineering, University of California, Berkeley, California 94720, USA; Innovative Genomics Institute, University of California, Berkeley, California 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA; Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA; Department of Chemistry, University of California, Berkeley, California 94720, USA; Gladstone Institute of Data Science and Biotechnology. Gladstone Institutes, San Francisco, California 94158, USA. Electronic address: doudna@berkeley.edu.
¹⁰ Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China; Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China. Electronic address: junjiegogoliu@tsinghua.edu.cn.

Abstract

A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.

Keywords: CRISPR; Cas12e; CasX; DNA cleavage; RNA-guided DNA nuclease; cryo-EM; genome editing; nucleic acid manipulation; sgRNA; structural engineering.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems / genetics
Endonucleases / genetics
Endonucleases / metabolism
Gene Editing* / methods
Mammals / metabolism
RNA / genetics
RNA, Guide, CRISPR-Cas Systems* / genetics
RNA, Guide, CRISPR-Cas Systems* / metabolism

Substances

RNA, Guide, CRISPR-Cas Systems
RNA
Endonucleases

Abstract

Publication types

MeSH terms

Substances

Grants and funding