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bioRxiv · 10.1101/2022.02.21.481263

Closely related type II-C Cas9 orthologs recognize diverse PAMs

Wei, J.·Hou, L.·Liu, J.·Gao, S.·Qi, T.·Sun, S.·Wang, Y.
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Abstract

Autosomal dominant diseases can be treated by allele-specific disruption of mutant alleles. If the missense mutation generates a novel protospacer-adjacent motif (PAM), CRISPR-Cas9 can distinguish mutant alleles from wild-type alleles by a PAM-specific approach. Therefore, it is crucial to develop a CRISPR toolbox capable of recognizing multiple PAMs. Here, by 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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Wei, J., Hou, L., Liu, J., Gao, S., Qi, T., Sun, S., Wang, Y.. 2022-02-21. Closely related type II-C Cas9 orthologs recognize diverse PAMs. https://doi.org/10.1101/2022.02.21.481263

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