Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

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• dc.contributor.author Alonso-Lerma, Borja
• dc.contributor.author Güell Cargol, Marc, 1982-
• dc.contributor.author Perez-Jimenez, Raul
• dc.date.accessioned 2023-09-28T12:06:47Z
• dc.date.available 2023-09-28T12:06:47Z
• dc.date.issued 2023
• dc.description.abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.
• dc.description.sponsorship This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494. We thank H. Stutzman for assistance with cloning plasmids, and Z. Herbert and M. Berkeley from the Molecular Biology Core Facilities at the Dana-Farber Cancer Institute for assistance with NextSeq sequencing.
• dc.format.mimetype application/pdf
• dc.identifier.citation Alonso-Lerma B, Jabalera Y, Samperio S, Morin M, Fernandez A, Hille LT, et al. Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins. Nat Microbiol. 2023 Jan;8(1):77-90. DOI: 10.1038/s41564-022-01265-y
• dc.identifier.doi http://dx.doi.org/10.1038/s41564-022-01265-y
• dc.identifier.issn 2058-5276
• dc.identifier.uri http://hdl.handle.net/10230/57987
• dc.language.iso eng
• dc.publisher Nature Research
• dc.relation.ispartof Nat Microbiol. 2023 Jan;8(1):77-90
• dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/964764
• dc.relation.projectID info:eu-repo/grantAgreement/ES/2PE/PID2019-109087RB-100
• dc.relation.projectID info:eu-repo/grantAgreement/ES/2PE/RTI2018-101223-B-100
• dc.relation.projectID info:eu-repo/grantAgreement/ES/3PE/PID2021-127644OB-100
• dc.rights © Springer Nature Publishing AG [Alonso-Lerma B, Jabalera Y, Samperio S, Morin M, Fernandez A, Hille LT, et al. Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins. Nat Microbiol. 2023 Jan;8(1):77-90. DOI: 10.1038/s41564-022-01265-y] [http://dx.doi.org/10.1038/s41564-022-01265-y]
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.subject.keyword Molecular evolution
• dc.subject.keyword Synthetic biology
• dc.title Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/acceptedVersion