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Nat Biotechnol DOI:10.1038/s41587-022-01410-2

High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs.

Publication TypeJournal Article
Year of Publication2022
AuthorsHuang, TP, Heins, ZJ, Miller, SM, Wong, BG, Balivada, PA, Wang, T, Khalil, AS, Liu, DR
JournalNat Biotechnol
Date Published2022 Sep 08
ISSN1546-1696
Abstract

Despite the availability of Cas9 variants with varied protospacer-adjacent motif (PAM) compatibilities, some genomic loci-especially those with pyrimidine-rich PAM sequences-remain inaccessible by high-activity Cas9 proteins. Moreover, broadening PAM sequence compatibility through engineering can increase off-target activity. With directed evolution, we generated four Cas9 variants that together enable targeting of most pyrimidine-rich PAM sequences in the human genome. Using phage-assisted noncontinuous evolution and eVOLVER-supported phage-assisted continuous evolution, we evolved Nme2Cas9, a compact Cas9 variant, into variants that recognize single-nucleotide pyrimidine-PAM sequences. We developed a general selection strategy that requires functional editing with fully specified target protospacers and PAMs. We applied this selection to evolve high-activity variants eNme2-T.1, eNme2-T.2, eNme2-C and eNme2-C.NR. Variants eNme2-T.1 and eNme2-T.2 offer access to NTN PAM sequences with comparable editing efficiencies as existing variants, while eNme2-C and eNme2-C.NR offer less restrictive PAM requirements, comparable or higher activity in a variety of human cell types and lower off-target activity at NCN PAM sequences.

DOI10.1038/s41587-022-01410-2
Pubmed

https://www.ncbi.nlm.nih.gov/pubmed/36076084?dopt=Abstract

Alternate JournalNat Biotechnol
PubMed ID36076084
Grant ListR01EB027793 / / U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB) /
R01EB031172 / / U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB) /
U01AI142756 / / U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID) /
R35GM118062 / / U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS) /
RM1HG009490 / / U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI) /
Liu investigatorship / / Howard Hughes Medical Institute (HHMI) /