Journal
NATURE COMMUNICATIONS
Volume 13, Issue 1, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41467-022-30228-4
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Funding
- Ministerio de Ciencia, Innovacion y Universidades, Agencia Estatal de Investigacion (AEI), through the Retos grant [PID2020-114986RB-100]
- Spanish Ministerio de Ciencia, Innovacion y Universidades [RyC-2017-23041, MDM-2016-0687, PGC2018-097260-B-I00]
- Universidad Pablo de Olavide (UPO)
- Universidad Pablo de Olavide (UPO) from CABD
- Fondo Europeo de Desarrollo Regional (FEDER)
- Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades de la Junta de Andalucia, within the operative program FEDER Andalucia 2014-2020 [01, P20_00866]
- European Union [PGC2018-097260-B-I00]
- Pablo de Olavide University
- Consejo Superior de Investigaciones Cientificas (CSIC)
- Junta de Andalucia
- Ayudas Puente Predoctorales (Universidad Pablo de Olavide, V Plan Propio de Investigacion y transferencia)
- la Caixa Foundation - Marie Skodowska-Curie grant [LCF/BQ/IN17/11620065, 713673]
- FI AGAUR fellowship from Generalitat de Catalunya [2018FI_B1_00511]
- National Institutes of Health (NIH) [R00 CA218870, P01 HL142494, 5R35GM122580-05]
- Marie Curie Actions (MSCA) [713673] Funding Source: Marie Curie Actions (MSCA)
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The authors validated and optimized the use of SpG and SpRY in vivo, expanding the targeting possibilities in animal genomes.
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.
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