Huang, Tony P.Heins, Zachary J.Miller, Shannon M.Wong, Brandon G.Balivada, Pallavi A.Wang, TinaKhalil, Ahmad S.Liu, David R.Liu, David2023-11-062022-09-08Huang, Tony P., Zachary J. Heins, Shannon M. Miller, Brandon G. Wong, Pallavi A. Balivada, Tina Wang, Ahmad S. Khalil et al. "High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs." Nat Biotechnol 41, no. 1 (2022): 96-107. DOI: 10.1038/s41587-022-01410-21087-01561546-1696https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37377346Despite 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 N4TN 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 N4CN PAM sequences.en-USBiomedical EngineeringMolecular MedicineApplied Microbiology and BiotechnologyBioengineeringBiotechnologyJournal Article2023-11-0610.1038/s41587-022-01410-2