High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage
Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISP...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier BV
2020
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| Online Access: | https://hdl.handle.net/1721.1/126488 |
| _version_ | 1826215163187953664 |
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| author | Slaymaker, Ian Mesa, Pablo Kellner, Max J. Kannan, Soumya Brignole, Edward J Koob, Jeremy Feliciano, Patricia Rosa Stella, Stefano Abudayyeh, Omar O. Gootenberg, Jonathan S Strecker, Jonathan Montoya, Guillermo Zhang, Feng |
| author2 | McGovern Institute for Brain Research at MIT |
| author_facet | McGovern Institute for Brain Research at MIT Slaymaker, Ian Mesa, Pablo Kellner, Max J. Kannan, Soumya Brignole, Edward J Koob, Jeremy Feliciano, Patricia Rosa Stella, Stefano Abudayyeh, Omar O. Gootenberg, Jonathan S Strecker, Jonathan Montoya, Guillermo Zhang, Feng |
| author_sort | Slaymaker, Ian |
| collection | MIT |
| description | Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells. Slaymaker et al. present a high-resolution structure of Cas13b in combination with biochemical studies showing that Cas13b is a highly dynamic, multi-turnover enzyme distinct from other Cas13 family members. Additionally, the authors demonstrate Cas13b can be rationally engineered to change RNA cleavage specificity and create a minimal variant suitable for delivery with viral vectors. |
| first_indexed | 2024-09-23T16:18:09Z |
| format | Article |
| id | mit-1721.1/126488 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T16:18:09Z |
| publishDate | 2020 |
| publisher | Elsevier BV |
| record_format | dspace |
| spelling | mit-1721.1/1264882022-09-29T19:27:52Z High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage Slaymaker, Ian Mesa, Pablo Kellner, Max J. Kannan, Soumya Brignole, Edward J Koob, Jeremy Feliciano, Patricia Rosa Stella, Stefano Abudayyeh, Omar O. Gootenberg, Jonathan S Strecker, Jonathan Montoya, Guillermo Zhang, Feng McGovern Institute for Brain Research at MIT Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Biology Harvard University--MIT Division of Health Sciences and Technology Broad Institute of MIT and Harvard Type VI CRISPR-Cas systems contain programmable single-effector RNA-guided RNases, including Cas13b, one of the four known family members. Cas13b, which has been used for both RNA editing and nucleic acid detection, is unique among type VI CRISPR effectors in its linear domain architecture and CRISPR RNA (crRNA) structure. Here, we report the crystal structure of Prevotella buccae Cas13b (PbuCas13b) bound to crRNA at 1.65 Å resolution. This structure, combined with biochemical experiments assaying the stability, kinetics, and function of Cas13b, provides a mechanistic model for Cas13b target RNA recognition and identifies features responsible for target and cleavage specificity. Based on these observations, we generated Cas13b variants with altered cleavage preferences, which may expand the utility of nuclease-based RNA detection assays and other applications of Cas13b in mammalian cells. Slaymaker et al. present a high-resolution structure of Cas13b in combination with biochemical studies showing that Cas13b is a highly dynamic, multi-turnover enzyme distinct from other Cas13 family members. Additionally, the authors demonstrate Cas13b can be rationally engineered to change RNA cleavage specificity and create a minimal variant suitable for delivery with viral vectors. NIH (Grants F30-NRSA-1F30-CA210382, 1R01-HG009761, 1R01-MH110049 and 1DP1-HL141201) 2020-08-06T17:56:31Z 2020-08-06T17:56:31Z 2019-03 2019-01 2019-10-08T13:20:50Z Article http://purl.org/eprint/type/JournalArticle 2211-1247 https://hdl.handle.net/1721.1/126488 Slaymaker, Ian M. et al. "High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage." Cell Reports 26, 16 (March 2019): P3741-3751.e5 © 2019 The Author(s) en http://dx.doi.org/10.1016/j.celrep.2019.02.094 Cell Reports Creative Commons Attribution 4.0 International license https://creativecommons.org/licenses/by/4.0/ application/pdf Elsevier BV Elsevier |
| spellingShingle | Slaymaker, Ian Mesa, Pablo Kellner, Max J. Kannan, Soumya Brignole, Edward J Koob, Jeremy Feliciano, Patricia Rosa Stella, Stefano Abudayyeh, Omar O. Gootenberg, Jonathan S Strecker, Jonathan Montoya, Guillermo Zhang, Feng High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage |
| title | High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage |
| title_full | High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage |
| title_fullStr | High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage |
| title_full_unstemmed | High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage |
| title_short | High-Resolution Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage |
| title_sort | high resolution structure of cas13b and biochemical characterization of rna targeting and cleavage |
| url | https://hdl.handle.net/1721.1/126488 |
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