Vicencio, Jeremy, 1990-Sánchez-Bolaños, CarlosMoreno-Sánchez, IsmaelBrena, DavidVejnar, Charles E.Kukhtar, DmytroRuiz-López, MiguelCots-Ponjoan, MarionaRubio, AlejandroRodrigo Melero, NataliaCrespo-Cuadrado, JesúsCarolis, CarloPérez-Pulido, Antonio J.Giráldez, AntonioKleinstiver, Benjamin P.Cerón Madrigal, JuliánMoreno Mateos, Miguel Ángel2022-06-212022-06-212022Vicencio 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/s41467-022-30228-42041-1723http://hdl.handle.net/10230/53553The 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.application/pdfeng© 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 madeGenòmicaGenèticaEnzimsPeix zebraCaenorhabditis elegansinfo:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-022-30228-4info:eu-repo/semantics/openAccess