DNA targeting by compact Cas9d and its resurrected ancestor
Abstract Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in...
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Nature Portfolio
2025-01-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-55573-4 |
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| author | Rodrigo Fregoso Ocampo Jack P. K. Bravo Tyler L. Dangerfield Isabel Nocedal Samatar A. Jirde Lisa M. Alexander Nicole C. Thomas Anjali Das Sarah Nielson Kenneth A. Johnson Christopher T. Brown Cristina N. Butterfield Daniela S. A. Goltsman David W. Taylor |
| author_facet | Rodrigo Fregoso Ocampo Jack P. K. Bravo Tyler L. Dangerfield Isabel Nocedal Samatar A. Jirde Lisa M. Alexander Nicole C. Thomas Anjali Das Sarah Nielson Kenneth A. Johnson Christopher T. Brown Cristina N. Butterfield Daniela S. A. Goltsman David W. Taylor |
| author_sort | Rodrigo Fregoso Ocampo |
| collection | DOAJ |
| description | Abstract Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases. |
| format | Article |
| id | doaj-art-6f097b25b0c7453cb917a5a4b8659965 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-6f097b25b0c7453cb917a5a4b86599652025-01-12T12:30:07ZengNature PortfolioNature Communications2041-17232025-01-0116111610.1038/s41467-024-55573-4DNA targeting by compact Cas9d and its resurrected ancestorRodrigo Fregoso Ocampo0Jack P. K. Bravo1Tyler L. Dangerfield2Isabel Nocedal3Samatar A. Jirde4Lisa M. Alexander5Nicole C. Thomas6Anjali Das7Sarah Nielson8Kenneth A. Johnson9Christopher T. Brown10Cristina N. Butterfield11Daniela S. A. Goltsman12David W. Taylor13Interdisciplinary Life Sciences Graduate Programs, University of Texas at AustinDepartment of Molecular Biosciences, University of Texas at AustinDepartment of Molecular Biosciences, University of Texas at AustinMetagenomi, Inc.Metagenomi, Inc.Metagenomi, Inc.Metagenomi, Inc.Department of Molecular Biosciences, University of Texas at AustinInterdisciplinary Life Sciences Graduate Programs, University of Texas at AustinInterdisciplinary Life Sciences Graduate Programs, University of Texas at AustinMetagenomi, Inc.Metagenomi, Inc.Metagenomi, Inc.Interdisciplinary Life Sciences Graduate Programs, University of Texas at AustinAbstract Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases.https://doi.org/10.1038/s41467-024-55573-4 |
| spellingShingle | Rodrigo Fregoso Ocampo Jack P. K. Bravo Tyler L. Dangerfield Isabel Nocedal Samatar A. Jirde Lisa M. Alexander Nicole C. Thomas Anjali Das Sarah Nielson Kenneth A. Johnson Christopher T. Brown Cristina N. Butterfield Daniela S. A. Goltsman David W. Taylor DNA targeting by compact Cas9d and its resurrected ancestor Nature Communications |
| title | DNA targeting by compact Cas9d and its resurrected ancestor |
| title_full | DNA targeting by compact Cas9d and its resurrected ancestor |
| title_fullStr | DNA targeting by compact Cas9d and its resurrected ancestor |
| title_full_unstemmed | DNA targeting by compact Cas9d and its resurrected ancestor |
| title_short | DNA targeting by compact Cas9d and its resurrected ancestor |
| title_sort | dna targeting by compact cas9d and its resurrected ancestor |
| url | https://doi.org/10.1038/s41467-024-55573-4 |
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