Minimal PAM specificity of a highly similar SpCas9 ortholog
RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetabl...
| Main Authors: | , , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
American Association for the Advancement of Science (AAAS)
2020
|
| Online Access: | https://hdl.handle.net/1721.1/126805 |
| _version_ | 1811090187720065024 |
|---|---|
| author | Chatterjee, Pranam Jakimo, Noah Jacobson, Joseph |
| author2 | Massachusetts Institute of Technology. Center for Bits and Atoms |
| author_facet | Massachusetts Institute of Technology. Center for Bits and Atoms Chatterjee, Pranam Jakimo, Noah Jacobson, Joseph |
| author_sort | Chatterjee, Pranam |
| collection | MIT |
| description | RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetable sequence space. In this study, we demonstrate the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report affinity to minimal 5′-NNG-3′ PAM sequences and demonstrate the accurate editing capabilities of the ortholog in both bacterial and human cells. Last, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be used both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities. ©2018 The Authors, some rights reserved. |
| first_indexed | 2024-09-23T14:37:07Z |
| format | Article |
| id | mit-1721.1/126805 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T14:37:07Z |
| publishDate | 2020 |
| publisher | American Association for the Advancement of Science (AAAS) |
| record_format | dspace |
| spelling | mit-1721.1/1268052022-09-29T09:58:55Z Minimal PAM specificity of a highly similar SpCas9 ortholog Chatterjee, Pranam Jakimo, Noah Jacobson, Joseph Massachusetts Institute of Technology. Center for Bits and Atoms Massachusetts Institute of Technology. Media Laboratory RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetable sequence space. In this study, we demonstrate the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report affinity to minimal 5′-NNG-3′ PAM sequences and demonstrate the accurate editing capabilities of the ortholog in both bacterial and human cells. Last, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be used both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities. ©2018 The Authors, some rights reserved. 2020-08-25T19:18:58Z 2020-08-25T19:18:58Z 2018-10 2018-05 2019-07-23T14:27:51Z Article http://purl.org/eprint/type/JournalArticle 2375-2548 https://hdl.handle.net/1721.1/126805 Chatterjee, Pranam et al., "Minimal PAM specificity of a highly similar SpCas9 ortholog." Science Advances 4, 10 (October 2018): no. eaau0766 doi. 10.1126/sciadv.aau0766 ©2018 Authors en https://dx.doi.org/10.1126/SCIADV.AAU0766 Science Advances Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf American Association for the Advancement of Science (AAAS) Science Advances |
| spellingShingle | Chatterjee, Pranam Jakimo, Noah Jacobson, Joseph Minimal PAM specificity of a highly similar SpCas9 ortholog |
| title | Minimal PAM specificity of a highly similar SpCas9 ortholog |
| title_full | Minimal PAM specificity of a highly similar SpCas9 ortholog |
| title_fullStr | Minimal PAM specificity of a highly similar SpCas9 ortholog |
| title_full_unstemmed | Minimal PAM specificity of a highly similar SpCas9 ortholog |
| title_short | Minimal PAM specificity of a highly similar SpCas9 ortholog |
| title_sort | minimal pam specificity of a highly similar spcas9 ortholog |
| url | https://hdl.handle.net/1721.1/126805 |
| work_keys_str_mv | AT chatterjeepranam minimalpamspecificityofahighlysimilarspcas9ortholog AT jakimonoah minimalpamspecificityofahighlysimilarspcas9ortholog AT jacobsonjoseph minimalpamspecificityofahighlysimilarspcas9ortholog |