Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

Vicencio, Jeremy, 1990-; Sánchez-Bolaños, Carlos; Moreno-Sánchez, Ismael; Brena, David; Vejnar, Charles E.; Kukhtar, Dmytro; Ruiz-López, Miguel; Cots-Ponjoan, Mariona; Rubio, Alejandro; Rodrigo Melero, Natalia; Crespo-Cuadrado, Jesús; Carolis, Carlo; Pérez-Pulido, Antonio J.; Giráldez, Antonio; Kleinstiver, Benjamin P.; Cerón Madrigal, Julián; Moreno Mateos, Miguel Ángel

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

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dc.contributor.author Vicencio, Jeremy, 1990-
dc.contributor.author Sánchez-Bolaños, Carlos
dc.contributor.author Moreno-Sánchez, Ismael
dc.contributor.author Brena, David
dc.contributor.author Vejnar, Charles E.
dc.contributor.author Kukhtar, Dmytro
dc.contributor.author Ruiz-López, Miguel
dc.contributor.author Cots-Ponjoan, Mariona
dc.contributor.author Rubio, Alejandro
dc.contributor.author Rodrigo Melero, Natalia
dc.contributor.author Crespo-Cuadrado, Jesús
dc.contributor.author Carolis, Carlo
dc.contributor.author Pérez-Pulido, Antonio J.
dc.contributor.author Giráldez, Antonio
dc.contributor.author Kleinstiver, Benjamin P.
dc.contributor.author Cerón Madrigal, Julián
dc.contributor.author Moreno Mateos, Miguel Ángel
dc.date.accessioned 2022-06-21T09:17:58Z
dc.date.available 2022-06-21T09:17:58Z
dc.date.issued 2022
dc.description.abstract The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals. Here we study the nuclease activity of SpG and SpRY by targeting 40 sites in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm could help to predict SpG and SpRY targets with high activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.
dc.description.sponsorship This study at Cerón Lab has been funded by Ministerio de Ciencia, Innovación y Universidades, which is part of Agencia Estatal de Investigación (AEI), through the Retos grant, number PID2020-114986RB-100. This work was also supported by Ramon y Cajal (RyC-2017-23041) and MDM-2016-0687 programs (Spanish Ministerio de Ciencia, Innovación y Universidades), Universidad Pablo de Olavide (UPO) Research and the Springboard programs from UPO and CABD, respectively and has been co-funded by the Fondo Europeo de Desarrollo Regional (FEDER) and Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía, within the operative program FEDER Andalucía 2014-2020 (01 - Refuerzo de la investigación, el desarrollo tecnológico y la innovación, grant P20_00866) (M.A.M.-M.). The Moreno-Mateos lab is supported by PGC2018-097260-B-I00 grant from Spanish Ministerio de Ciencia, Innovación y Universidades and European Union. J.V. had an INPhINIT PhD fellowship from “la Caixa” Foundation (LCF/BQ/IN17/11620065) co-funded by Marie Skłodowska-Curie grant agreement no. 713673. D.B. is a predoctoral fellow of the CONACYT “Becas al Extranjero” Program of Mexico. D.K. has an FI AGAUR fellowship (2018FI_B1_00511) from Generalitat de Catalunya. A.J.G. and B.P.K. acknowledge support from National Institutes of Health (NIH) R00 CA218870 and NIH P01 HL142494 (B.P.K.) and 5R35GM122580-05 (A.J.G.)
dc.format.mimetype application/pdf
dc.identifier.citation Vicencio J, Sánchez-Bolaños C, Moreno-Sánchez I, Brena D, Vejnar CE, Kukhtar D et al. Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes. Nat Commun. 2022 May 12;13(1):2601. DOI:10.1038/s41467-022-30228-4
dc.identifier.doi http://dx.doi.org/10.1038/s41467-022-30228-4
dc.identifier.issn 2041-1723
dc.identifier.uri http://hdl.handle.net/10230/53553
dc.language.iso eng
dc.publisher Nature Research
dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/713673
dc.rights © Jeremy Vicencio et al. 2022. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made
dc.rights.accessRights info:eu-repo/semantics/openAccess
dc.rights.uri https://creativecommons.org/licenses/by/4.0/
dc.subject.other Genòmica
dc.subject.other Genètica
dc.subject.other Enzims
dc.subject.other Peix zebra
dc.subject.other Caenorhabditis elegans
dc.title Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
dc.type info:eu-repo/semantics/article
dc.type.version info:eu-repo/semantics/publishedVersion

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