Please use this identifier to cite or link to this item:
http://hdl.handle.net/2445/186096
Title: | Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes |
Author: | Vicencio, Jeremy 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 J. Kleinstiver, Benjamin P. Cerón, Julián Moreno Mateos, Miguel A. |
Keywords: | RNA Metabolisme RNA Metabolism |
Issue Date: | 12-May-2022 |
Publisher: | Springer Science and Business Media |
Abstract: | PAM requirement is a constraint for genome editing but this has been circumvented by engineered Cas9 nucleases as SpG and SpRY recognizing minimal PAM sequences. Here, the authors validate and optimize SpG and SpRY in vivo expanding the targeting landscape in animals. 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. |
Note: | Reproducció del document publicat a: https://doi.org/10.1038/s41467-022-30228-4 |
It is part of: | Nature Communications, 2022, vol. 13, num. 1 |
URI: | http://hdl.handle.net/2445/186096 |
Related resource: | https://doi.org/10.1038/s41467-022-30228-4 |
ISSN: | 2041-1723 |
Appears in Collections: | Articles publicats en revistes (Institut d'lnvestigació Biomèdica de Bellvitge (IDIBELL)) |
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File | Description | Size | Format | |
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s41467-022-30228-4.pdf | 3.25 MB | Adobe PDF | View/Open |
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