Closely related type II-C Cas9 orthologs recognize diverse PAMs
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.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file.
Article and author information
Author details
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.
Reviewing Editor
- Jeremy J Day, University of Alabama at Birmingham, United States
Version history
- Received: February 11, 2022
- Preprint posted: February 21, 2022 (view preprint)
- Accepted: August 11, 2022
- Accepted Manuscript published: August 12, 2022 (version 1)
- Version of Record published: August 31, 2022 (version 2)
Copyright
© 2022, Wei et al.
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.
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