High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs
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 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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Sep 08, 2022
- Source ID
- 10.1038/s41587-022-01410-2
Entities
People
- Ahmad S Khalil
- Brandon G. Wong
- David R. Liu
- Pallavi A. Balivada
- Shannon M. Miller
- Tina Wang
- Tony P. Huang
- Zachary Heins
Organizations
- Howard Hughes Medical Institute
- National Human Genome Research Institute
- National Institute of Allergy and Infectious Diseases
- National Institute of Biomedical Imaging and Bioengineering
- National Institute of General Medical Sciences