Cargando…
Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition
Due to different nucleotide preferences at target sites, no single Cas9 is capable of editing all sequences. Thus, this highlights the need to establish a Cas9 repertoire covering all sequences for efficient genome editing. Cas9s with simple protospacer adjacent motif (PAM) requirements are particul...
Autores principales: | , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9710800/ https://www.ncbi.nlm.nih.gov/pubmed/36449487 http://dx.doi.org/10.1371/journal.pbio.3001897 |
_version_ | 1784841445774983168 |
---|---|
author | Wang, Shuai Tao, Chen Mao, Huilin Hou, Linghui Wang, Yao Qi, Tao Yang, Yuan Ong, Sang-Ging Hu, Shijun Chai, Renjie Wang, Yongming |
author_facet | Wang, Shuai Tao, Chen Mao, Huilin Hou, Linghui Wang, Yao Qi, Tao Yang, Yuan Ong, Sang-Ging Hu, Shijun Chai, Renjie Wang, Yongming |
author_sort | Wang, Shuai |
collection | PubMed |
description | Due to different nucleotide preferences at target sites, no single Cas9 is capable of editing all sequences. Thus, this highlights the need to establish a Cas9 repertoire covering all sequences for efficient genome editing. Cas9s with simple protospacer adjacent motif (PAM) requirements are particularly attractive to allow for a wide range of genome editing, but identification of such Cas9s from thousands of Cas9s in the public database is a challenge. We previously identified PAMs for 16 SaCas9 orthologs. Here, we compared the PAM-interacting (PI) domains in these orthologs and found that the serine residue corresponding to SaCas9 N986 was associated with the simple NNGG PAM requirement. Based on this discovery, we identified five additional SaCas9 orthologs that recognize the NNGG PAM. We further identified three amino acids that determined the NNGG PAM requirement of SaCas9. Finally, we engineered Sha2Cas9 and SpeCas9 to generate high-fidelity versions of Cas9s. Importantly, these natural and engineered Cas9s displayed high activities and distinct nucleotide preferences. Our study offers a new perspective to identify SaCas9 orthologs with NNGG PAM requirements, expanding the Cas9 repertoire. |
format | Online Article Text |
id | pubmed-9710800 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-97108002022-12-01 Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition Wang, Shuai Tao, Chen Mao, Huilin Hou, Linghui Wang, Yao Qi, Tao Yang, Yuan Ong, Sang-Ging Hu, Shijun Chai, Renjie Wang, Yongming PLoS Biol Research Article Due to different nucleotide preferences at target sites, no single Cas9 is capable of editing all sequences. Thus, this highlights the need to establish a Cas9 repertoire covering all sequences for efficient genome editing. Cas9s with simple protospacer adjacent motif (PAM) requirements are particularly attractive to allow for a wide range of genome editing, but identification of such Cas9s from thousands of Cas9s in the public database is a challenge. We previously identified PAMs for 16 SaCas9 orthologs. Here, we compared the PAM-interacting (PI) domains in these orthologs and found that the serine residue corresponding to SaCas9 N986 was associated with the simple NNGG PAM requirement. Based on this discovery, we identified five additional SaCas9 orthologs that recognize the NNGG PAM. We further identified three amino acids that determined the NNGG PAM requirement of SaCas9. Finally, we engineered Sha2Cas9 and SpeCas9 to generate high-fidelity versions of Cas9s. Importantly, these natural and engineered Cas9s displayed high activities and distinct nucleotide preferences. Our study offers a new perspective to identify SaCas9 orthologs with NNGG PAM requirements, expanding the Cas9 repertoire. Public Library of Science 2022-11-30 /pmc/articles/PMC9710800/ /pubmed/36449487 http://dx.doi.org/10.1371/journal.pbio.3001897 Text en © 2022 Wang et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Wang, Shuai Tao, Chen Mao, Huilin Hou, Linghui Wang, Yao Qi, Tao Yang, Yuan Ong, Sang-Ging Hu, Shijun Chai, Renjie Wang, Yongming Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition |
title | Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition |
title_full | Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition |
title_fullStr | Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition |
title_full_unstemmed | Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition |
title_short | Identification of SaCas9 orthologs containing a conserved serine residue that determines simple NNGG PAM recognition |
title_sort | identification of sacas9 orthologs containing a conserved serine residue that determines simple nngg pam recognition |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9710800/ https://www.ncbi.nlm.nih.gov/pubmed/36449487 http://dx.doi.org/10.1371/journal.pbio.3001897 |
work_keys_str_mv | AT wangshuai identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT taochen identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT maohuilin identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT houlinghui identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT wangyao identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT qitao identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT yangyuan identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT ongsangging identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT hushijun identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT chairenjie identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition AT wangyongming identificationofsacas9orthologscontainingaconservedserineresiduethatdeterminessimplennggpamrecognition |