Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

Jeremy Vicencio, Carlos Sánchez-Bolaños, Ismael Moreno-Sánchez, David Brena, Charles E. Vejnar, Dmytro Kukhtar, Miguel Ruiz-López, Mariona Cots-Ponjoan, Alejandro Rubio, Natalia Rodrigo Melero, Jesús Crespo-Cuadrado, Carlo Carolis, Antonio J. Pérez-Pulido, Antonio J. Giráldez, Benjamin P. Kleinstiver, Julián Cerón () and Miguel A. Moreno-Mateos ()
Additional contact information
Jeremy Vicencio: Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL
Carlos Sánchez-Bolaños: Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
Ismael Moreno-Sánchez: Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
David Brena: Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL
Charles E. Vejnar: Yale University School of Medicine
Dmytro Kukhtar: Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL
Miguel Ruiz-López: Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL
Mariona Cots-Ponjoan: Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL
Alejandro Rubio: Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
Natalia Rodrigo Melero: Biomolecular Screening and Protein Technologies Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology
Jesús Crespo-Cuadrado: Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
Carlo Carolis: Biomolecular Screening and Protein Technologies Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology
Antonio J. Pérez-Pulido: Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
Antonio J. Giráldez: Yale University School of Medicine
Benjamin P. Kleinstiver: Center for Genomic Medicine, Massachusetts General Hospital
Julián Cerón: Modeling human diseases in C. elegans Group; Genes, Disease and Therapy Program, Institut d’Investigació Biomèdica de Bellvitge - IDIBELL
Miguel A. Moreno-Mateos: Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-30228-4

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