RT Journal Article
T1 Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
A1 Vicencio, Jeremy
A1 Sánchez-Bolaños, Carlos
A1 Moreno-Sánchez, Ismael
A1 Brena, David
A1 Vejnar, Charles E.
A1 Kuthar, Dmytro
A1 Ruiz-López, Miguel
A1 Cots-Ponjoan, Mariona
A1 Rubio Valle, Alejandro
A1 Rodrigo Melero, Natalia
A1 Crespo Cuadrado, Jesús
A1 Carolis, Carlos
A1 Pérez-Pulido, Antonio J.
A1 Giraldez, Antonio J.
A1 Kleinstiver, Benjamin P.
A1 Cerón, Julián
A1 Moreno Mateos, Miguel A.
K1 CRISPR-Cas
K1 Genome editing
K1 Zebrafish
K1 Nematodes
AB 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.
PB Nature
YR 2022
FD 2022-05-12
LK https://hdl.handle.net/10433/26116
UL https://hdl.handle.net/10433/26116
LA en
NO Nature Communications, 13, 2601
NO Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía, Ctra. Utrera Km.1, 41013 Seville, Spain
NO Department of Molecular Biology and Biochemical Engineering, Pablo de Olavide University, Ctra. Utrera Km.1, 41013 Seville, Spain
DS RIO
RD May 22, 2026