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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.
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A bacterial dual positive and negative selection system for dCas9 activity
The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E . coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin. The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin. This system is capable of enriching for rare dCas9 variants up to 9,000-fold and possesses potential utility in directed evolution experiments to create switchable dCas9 proteins.
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- Award ID(s):
- 1803805
- PAR ID:
- 10339670
- Editor(s):
- Fujii, Hodaka
- Date Published:
- Journal Name:
- PLOS ONE
- Volume:
- 17
- Issue:
- 6
- ISSN:
- 1932-6203
- Page Range / eLocation ID:
- e0269270
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pone.0269270
