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A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9

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...

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Autores principales: Collias, D., Leenay, R. T., Slotkowski, R. A., Zuo, Z., Collins, S. P., McGirr, B. A., Liu, J., Beisel, C. L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Association for the Advancement of Science 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439565/
https://www.ncbi.nlm.nih.gov/pubmed/32832642
http://dx.doi.org/10.1126/sciadv.abb4054
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author Collias, D.
Leenay, R. T.
Slotkowski, R. A.
Zuo, Z.
Collins, S. P.
McGirr, B. A.
Liu, J.
Beisel, C. L.
author_facet Collias, D.
Leenay, R. T.
Slotkowski, R. A.
Zuo, Z.
Collins, S. P.
McGirr, B. A.
Liu, J.
Beisel, C. L.
author_sort Collias, D.
collection PubMed
description 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.
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spelling pubmed-74395652020-08-20 A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9 Collias, D. Leenay, R. T. Slotkowski, R. A. Zuo, Z. Collins, S. P. McGirr, B. A. Liu, J. Beisel, C. L. Sci Adv Research Articles 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. American Association for the Advancement of Science 2020-07-15 /pmc/articles/PMC7439565/ /pubmed/32832642 http://dx.doi.org/10.1126/sciadv.abb4054 Text en Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license (https://creativecommons.org/licenses/by-nc/4.0/) , which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.
spellingShingle Research Articles
Collias, D.
Leenay, R. T.
Slotkowski, R. A.
Zuo, Z.
Collins, S. P.
McGirr, B. A.
Liu, J.
Beisel, C. L.
A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9
title A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9
title_full A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9
title_fullStr A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9
title_full_unstemmed A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9
title_short A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9
title_sort positive, growth-based pam screen identifies noncanonical motifs recognized by the s. pyogenes cas9
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439565/
https://www.ncbi.nlm.nih.gov/pubmed/32832642
http://dx.doi.org/10.1126/sciadv.abb4054
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