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Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria
[Image: see text] The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding mo...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10510713/ https://www.ncbi.nlm.nih.gov/pubmed/37643152 http://dx.doi.org/10.1021/acssynbio.3c00366 |
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author | Teng, Yuxi Wang, Jian Jiang, Tian Zou, Yusong Yan, Yajun |
author_facet | Teng, Yuxi Wang, Jian Jiang, Tian Zou, Yusong Yan, Yajun |
author_sort | Teng, Yuxi |
collection | PubMed |
description | [Image: see text] The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from Streptococcus equinus HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction. |
format | Online Article Text |
id | pubmed-10510713 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-105107132023-09-21 Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria Teng, Yuxi Wang, Jian Jiang, Tian Zou, Yusong Yan, Yajun ACS Synth Biol [Image: see text] The RNA-guided Cas9 endonucleases have revolutionized gene editing and regulation, but their targeting scope is limited by the protospacer adjacent motif (PAM) requirement. The most extensively used SpCas9 from Streptococcus pyogenes recognizes the NGG PAM via an RxR PAM-binding motif within its PAM-interaction (PI) domain. To overcome the strict PAM requirement, we identified and characterized a Cas9 ortholog from Streptococcus equinus HC5 (SeHCas9) that shows high sequence identity with SpCas9 but harbors a different RxQ PAM-binding motif. Complete PAM profiling revealed that SeHCas9 recognized an NAG PAM and accommodated NKG and NAW PAMs. We investigated the PAM interaction mechanism by identifying the crucial role of R1336 within the RxQ motif in determining PAM specificity, as well as the essentiality of two conserved residues (R1152 and Q1229) across Cas9 orthologs bearing the RxQ motif for PAM recognition. Further protein engineering created two variants, SeHdCas9-Q1229R and SeHdCas9-RR, that showed robust repression across an NNG and NNN PAM range, respectively. Our work proposes a novel Cas9 PAM interaction mechanism and establishes PAM-free Cas9 variants for bacterial gene control with almost no targeting restriction. American Chemical Society 2023-08-29 /pmc/articles/PMC10510713/ /pubmed/37643152 http://dx.doi.org/10.1021/acssynbio.3c00366 Text en © 2023 American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Teng, Yuxi Wang, Jian Jiang, Tian Zou, Yusong Yan, Yajun Engineering a Streptococcus Cas9 Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in Bacteria |
title | Engineering a Streptococcus Cas9
Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in
Bacteria |
title_full | Engineering a Streptococcus Cas9
Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in
Bacteria |
title_fullStr | Engineering a Streptococcus Cas9
Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in
Bacteria |
title_full_unstemmed | Engineering a Streptococcus Cas9
Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in
Bacteria |
title_short | Engineering a Streptococcus Cas9
Ortholog with an RxQ PAM-Binding Motif for PAM-Free Gene Control in
Bacteria |
title_sort | engineering a streptococcus cas9
ortholog with an rxq pam-binding motif for pam-free gene control in
bacteria |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10510713/ https://www.ncbi.nlm.nih.gov/pubmed/37643152 http://dx.doi.org/10.1021/acssynbio.3c00366 |
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