Closely related type II-C Cas9 orthologs recognize diverse PAMs
- Fudan University, China
- Sun Yat-sen University Cancer Center, China
- Children's Hospital of Fudan University, China
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.
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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.
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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Closely related type II-C Cas9 orthologs recognize diverse PAMs
eLife 11:e77825.
https://doi.org/10.7554/eLife.77825
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