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dc.contributor.authorCollias, D
dc.contributor.authorLeenay, R T
dc.contributor.authorSlotkowski, R A
dc.contributor.authorZuo, Z
dc.contributor.authorCollins, S P
dc.contributor.authorMcGirr, B A
dc.contributor.authorLiu, J
dc.contributor.authorBeisel, C L
dc.date.accessioned2020-09-25T09:55:08Z
dc.date.available2020-09-25T09:55:08Z
dc.date.issued2020-07-15
dc.identifier.citationSci Adv. 2020 Jul 15;6(29):eabb4054. doi: 10.1126/sciadv.abb4054.en_US
dc.identifier.pmid32832642
dc.identifier.doi10.1126/sciadv.abb4054
dc.identifier.urihttp://hdl.handle.net/10033/622459
dc.description.abstractCRISPR 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.isoenen_US
dc.publisherAAASen_US
dc.rightsAttribution-NonCommercial-ShareAlike 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.titleA positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9.en_US
dc.typeArticleen_US
dc.identifier.eissn2375-2548
dc.contributor.departmentHIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.en_US
dc.identifier.journalScience advancesen_US
dc.source.volume6
dc.source.issue29
dc.source.beginpageeabb4054
dc.source.endpage
refterms.dateFOA2020-09-25T09:55:08Z
dc.source.journaltitleScience advances
dc.source.countryUnited States


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