Closely related type II-C Cas9 orthologs recognize diverse PAMs

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Abstract

The RNA-guided CRISPR/Cas9 system is a powerful tool for genome editing, but its targeting scope is limited by the protospacer-adjacent motif (PAM). To expand the target scope, it is crucial to develop a CRISPR toolbox capable of recognizing multiple PAMs. Here, using a GFP-activation assay, we tested the activities of 29 type II-C orthologs closely related to Nme1Cas9, 25 of which are active in human cells. These orthologs recognize diverse PAMs with variable length and nucleotide preference, including purine-rich, pyrimidine-rich, and mixed purine and pyrimidine PAMs. We characterized in depth the activity and specificity of Nsp2Cas9. We also generated a chimeric Cas9 nuclease that recognizes a simple N₄C PAM, representing the most relaxed PAM preference for compact Cas9s to date. These Cas9 nucleases significantly enhance our ability to perform allele-specific genome editing.

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All data generated or analysed during this study are included in the manuscript and supporting file.

Article and author information

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Jingjing Wei

State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Linghui Hou

State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Jingtong Liu

State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Ziwen Wang

State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, China

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1678-7624
Siqi Gao

State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Tao Qi

State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Song Gao

State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, China

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7427-6681
Shuna Sun

National Children's Medical Center, Children's Hospital of Fudan University, Shanghai, China

For correspondence
sun_shuna@fudan.edu.cn

Competing interests
The authors declare that no competing interests exist.
Yongming Wang

State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China

For correspondence
ymw@fudan.edu.cn

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8269-5296

Funding

National Key Research and Development Program of China (2021YFA0910602,2021YFC2701103)

Yongming Wang

National Natural Science Foundation of China (82070258,81870199)

Yongming Wang

Open Research Fund of State Key Laboratory of Genetic Engineering, Fudan University (No. SKLGE-2104)

Yongming Wang

Science and Technology ReSearch Program of Shanghai (19DZ2282100)

Yongming Wang

Natural Science Fund of Shanghai Science and Technology Commission (19ZR1406300)

Yongming Wang

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.