Enhanced RNA-targeting CRISPR-Cas technology in zebrafish
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.
Authors
- Manuel J. Muñoz (ORCID: https://orcid.org/0000-0002-0111-1541)
- Anthony J. Treichel (ORCID: https://orcid.org/0000-0002-4393-7034)
- Carlos Gómez-Marín (ORCID: https://orcid.org/0000-0001-9724-2919)
- Ismael Moreno-Sánchez (ORCID: https://orcid.org/0000-0001-5606-4028)
- M. Ángela Nieto (ORCID: https://orcid.org/0000-0002-3538-840X)
- Gopal Kushawah (ORCID: https://orcid.org/0000-0002-3608-1530)
- Alejandra Cano-Ruiz (ORCID: https://orcid.org/0000-0003-4462-9225)
- John Walker (ORCID: https://orcid.org/0000-0002-3544-7668)
- Pedro Manuel Martínez‐García (ORCID: https://orcid.org/0000-0002-2119-2465)
- Alejandro Díaz‐Moscoso (ORCID: https://orcid.org/0000-0001-8750-6415)
- Kevin Holden (ORCID: https://orcid.org/0000-0002-3091-4124)
- Anoja Perera (ORCID: https://orcid.org/0000-0003-3965-5293)
- Ariel Bazzini (ORCID: https://orcid.org/0000-0002-2251-5174)
- Miguel A. Moreno-Mateos (ORCID: https://orcid.org/0000-0001-5723-0569)
- Laura Tomás‐Gallardo (ORCID: https://orcid.org/0000-0001-9661-0521)
- Rhonda Egidy
- Luis Hernández-Huertas
- Joan Galcerán (ORCID: https://orcid.org/0000-0002-5247-2726)
- Gabriel da Silva Pescador
- Daniel Nahón-Cano
Institutions
- Stowers Institute for Medical Research (US)
- Instituto de Salud Carlos III (ES)
- Instituto de Neurociencias (ES)
- Synthego (United States) (US)
- Centre for Biomedical Network Research on Rare Diseases (ES)
- Centro Andaluz de Biología del Desarrollo (ES)
- Centro de Investigación Biomédica en Red (ES)
- University of Kansas Medical Center (US)
- Instituto de Investigaciones Químicas (ES)
- Junta de Andalucía (ES)
- Universidad Pablo de Olavide (ES)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-03-16
- DOI
- https://doi.org/10.1038/s41467-025-57792-9
- Citations
- 13
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 10.48
Funders
- Stowers Institute for Medical Research
- Ministerio de Ciencia, Innovación y Universidades
- Consejería de Transformación Económica, Industria, Conocimiento y Universidades
- European Commission
- Generalitat Valenciana
- Ministerio de Ciencia e Innovación
- Junta de Andalucía
- National Institutes of Health
- Consejo Superior de Investigaciones Científicas
- Instituto de Salud Carlos III
- European Regional Development Fund
- Agencia Estatal de Investigación
- European Social Fund