A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9.
dc.contributor.author | Collias, D | |
dc.contributor.author | Leenay, R T | |
dc.contributor.author | Slotkowski, R A | |
dc.contributor.author | Zuo, Z | |
dc.contributor.author | Collins, S P | |
dc.contributor.author | McGirr, B A | |
dc.contributor.author | Liu, J | |
dc.contributor.author | Beisel, C L | |
dc.date.accessioned | 2020-09-25T09:55:08Z | |
dc.date.available | 2020-09-25T09:55:08Z | |
dc.date.issued | 2020-07-15 | |
dc.identifier.citation | Sci Adv. 2020 Jul 15;6(29):eabb4054. doi: 10.1126/sciadv.abb4054. | en_US |
dc.identifier.pmid | 32832642 | |
dc.identifier.doi | 10.1126/sciadv.abb4054 | |
dc.identifier.uri | http://hdl.handle.net/10033/622459 | |
dc.description.abstract | CRISPR technologies have overwhelmingly relied on the Streptococcus pyogenes Cas9 (SpyCas9), with its consensus NGG and less preferred NAG and NGA protospacer-adjacent motifs (PAMs). Here, we report that SpyCas9 also recognizes sequences within an N(A/C/T)GG motif. These sequences were identified on the basis of preferential enrichment in a growth-based screen in Escherichia coli. DNA binding, cleavage, and editing assays in bacteria and human cells validated recognition, with activities paralleling those for NAG(A/C/T) PAMs and dependent on the first two PAM positions. Molecular-dynamics simulations and plasmid-clearance assays with mismatch-intolerant variants supported induced-fit recognition of an extended PAM by SpyCas9 rather than recognition of NGG with a bulged R-loop. Last, the editing location for SpyCas9-derived base editors could be shifted by one nucleotide by selecting between (C/T)GG and adjacent N(C/T)GG PAMs. SpyCas9 and its enhanced variants thus recognize a larger repertoire of PAMs, with implications for precise editing, off-target predictions, and CRISPR-based immunity. | en_US |
dc.language.iso | en | en_US |
dc.publisher | AAAS | en_US |
dc.rights | Attribution-NonCommercial-ShareAlike 4.0 International | * |
dc.rights.uri | http://creativecommons.org/licenses/by-nc-sa/4.0/ | * |
dc.title | A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9. | en_US |
dc.type | Article | en_US |
dc.identifier.eissn | 2375-2548 | |
dc.contributor.department | HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany. | en_US |
dc.identifier.journal | Science advances | en_US |
dc.source.volume | 6 | |
dc.source.issue | 29 | |
dc.source.beginpage | eabb4054 | |
dc.source.endpage | ||
refterms.dateFOA | 2020-09-25T09:55:08Z | |
dc.source.journaltitle | Science advances | |
dc.source.country | United States |
Files in this item
This item appears in the following Collection(s)
-
publications of the research group biology of synthetic RNA ([HIRI] RSYN) [22]
Examination and utilization of CRISPER-CAS systems