Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

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.
Ceron laboratory thanks the CERCA Program (Generalitat de Catalunya) for their institutional support, and Nicholas Stroustrup and Alberto Villanueva for sharing reagents and equipment, and Marta Artal and Antonio Miranda for allowing to share the information on the efficiency of the trx-1 and usp-48 endogenous reporters generation. We thank Erik Jorgensen for sharing the EG9615 strain. We thank Jordan Ward for hosting J.C. at the UCSC where cki-1 tagging was performed. 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. The CABD is an institution funded by Pablo de Olavide University, Consejo Superior de Investigaciones Científicas (CSIC), and Junta de Andalucía. C.S.-B. was a recipient of the Ayudas Puente Predoctorales (Universidad Pablo de Olavide, V Plan Propio de Investigación y transferencia). 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.).