Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

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Title: Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins
Authors: Alonso-Lerma, Borja | Jabalera, Ylenia | Samperio, Sara | Morin, Matias | Fernandez, Almudena | Hille, Logan T. | Silverstein, Rachel A. | Quesada-Ganuza, Ane | Reifs, Antonio | Fernández-Peñalver, Sergio | Benitez, Yolanda | Soletto, Lucia | Gavira, Jose A. | Diaz, Adrian | Vranken, Wim | Sanchez-Mejias, Avencia | Güell, Marc | Mojica, Francisco J.M. | Kleinstiver, Benjamin P. | Moreno-Pelayo, Miguel A. | Montoliu, Lluis | Perez-Jimenez, Raul
Research Group/s: Microbiología Molecular
Center, Department or Service: Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología | Universidad de Alicante. Instituto Multidisciplinar para el Estudio del Medio "Ramón Margalef"
Keywords: CRISPR | Cas9 | Ancient nucleases | Resurrected proteins
Issue Date: 2-Jan-2023
Publisher: Springer Nature
Citation: Nature Microbiology. 2023, 8: 77-90. https://doi.org/10.1038/s41564-022-01265-y
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.
Sponsor: 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.
URI: http://hdl.handle.net/10045/130786
ISSN: 2058-5276
DOI: 10.1038/s41564-022-01265-y
Language: eng
Type: info:eu-repo/semantics/article
Rights: © The Author(s), under exclusive licence to Springer Nature Limited 2023
Peer Review: si
Publisher version: https://doi.org/10.1038/s41564-022-01265-y
Appears in Collections:Research funded by the EU
INV - Microbiología Molecular - Artículos de Revistas

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