High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs.
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.
|Year of Publication||
2022 Sep 08
R01EB027793 / 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)