Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

Ismael Moreno-Sánchez, Luis Hernández-Huertas, Daniel Nahón-Cano, Pedro Manuel Martínez-García, Anthony J. Treichel, Carlos Gómez-Marin, Laura Tomás-Gallardo, Gabriel Silva Pescador, Gopal Kushawah, Rhonda Egidy, Anoja Perera, Alejandro Díaz-Moscoso, Alejandra Cano-Ruiz, John A. Walker, Manuel J. Muñoz, Kevin Holden, Joan Galcerán, M. Ángela Nieto, Ariel A. Bazzini and Miguel A. Moreno-Mateos ()
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Ismael Moreno-Sánchez: Pablo de Olavide University/CSIC/Junta de Andalucía
Luis Hernández-Huertas: Pablo de Olavide University/CSIC/Junta de Andalucía
Daniel Nahón-Cano: Pablo de Olavide University/CSIC/Junta de Andalucía
Pedro Manuel Martínez-García: Pablo de Olavide University/CSIC/Junta de Andalucía
Anthony J. Treichel: Stowers Institute for Medical Research
Carlos Gómez-Marin: Pablo de Olavide University/CSIC/Junta de Andalucía
Laura Tomás-Gallardo: Pablo de Olavide University/CSIC/Junta de Andalucía
Gabriel Silva Pescador: Stowers Institute for Medical Research
Gopal Kushawah: Stowers Institute for Medical Research
Rhonda Egidy: Stowers Institute for Medical Research
Anoja Perera: Stowers Institute for Medical Research
Alejandro Díaz-Moscoso: Pablo de Olavide University/CSIC/Junta de Andalucía
Alejandra Cano-Ruiz: Pablo de Olavide University/CSIC/Junta de Andalucía
John A. Walker: Synthego Corporation
Manuel J. Muñoz: Pablo de Olavide University/CSIC/Junta de Andalucía
Kevin Holden: Synthego Corporation
Joan Galcerán: Instituto de Neurociencias (CSIC-UMH)
M. Ángela Nieto: Instituto de Neurociencias (CSIC-UMH)
Ariel A. Bazzini: Stowers Institute for Medical Research
Miguel A. Moreno-Mateos: Pablo de Olavide University/CSIC/Junta de Andalucía

Nature Communications, 2025, vol. 16, issue 1, 1-20

Abstract: Abstract CRISPR-Cas13 RNA-targeting systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its competence could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) use chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improve nuclear RNA targeting, and iii) compare different computational models and determine the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrate that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implement alternative RNA-targeting CRISPR-Cas systems such as CRISPR-Cas7-11 and CRISPR-DjCas13d. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

Date: 2025
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DOI: 10.1038/s41467-025-57792-9

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