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Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

Author

Listed:
  • Ismael Moreno-Sánchez

    (Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University
    Instituto de Neurociencias (CSIC-UMH))

  • Luis Hernández-Huertas

    (Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

  • Daniel Nahón-Cano

    (Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

  • 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
    Pablo de Olavide University)

  • Laura Tomás-Gallardo

    (Pablo de Olavide University/CSIC/Junta de Andalucía
    Andalusian Center for Developmental Biology (CABD), 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
    Andalusian Center for Developmental Biology (CABD), Pablo de Olavide University/CSIC/Junta de Andalucía
    CSIC-US)

  • Alejandra Cano-Ruiz

    (Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

  • John A. Walker

    (Synthego Corporation)

  • Manuel J. Muñoz

    (Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

  • Kevin Holden

    (Synthego Corporation)

  • Joan Galcerán

    (Instituto de Neurociencias (CSIC-UMH)
    ISCIII)

  • M. Ángela Nieto

    (Instituto de Neurociencias (CSIC-UMH)
    ISCIII)

  • Ariel A. Bazzini

    (Stowers Institute for Medical Research
    University of Kansas Medical Center)

  • Miguel A. Moreno-Mateos

    (Pablo de Olavide University/CSIC/Junta de Andalucía
    Pablo de Olavide University)

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.

Suggested Citation

  • 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 Kus, 2025. "Enhanced RNA-targeting CRISPR-Cas technology in zebrafish," Nature Communications, Nature, vol. 16(1), pages 1-20, December.
  • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-57792-9
    DOI: 10.1038/s41467-025-57792-9
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    References listed on IDEAS

    as
    1. Yage Ding & Cristina Tous & Jaehoon Choi & Jingyao Chen & Wilson W. Wong, 2024. "Orthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    2. Xiaolong Cheng & Zexu Li & Ruocheng Shan & Zihan Li & Shengnan Wang & Wenchang Zhao & Han Zhang & Lumen Chao & Jian Peng & Teng Fei & Wei Li, 2023. "Modeling CRISPR-Cas13d on-target and off-target effects using machine learning approaches," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    3. Alexander J. Meeske & Sandra Nakandakari-Higa & Luciano A. Marraffini, 2019. "Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage," Nature, Nature, vol. 570(7760), pages 241-245, June.
    4. Alexandra East-Seletsky & Mitchell R. O’Connell & Spencer C. Knight & David Burstein & Jamie H. D. Cate & Robert Tjian & Jennifer A. Doudna, 2016. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection," Nature, Nature, vol. 538(7624), pages 270-273, October.
    5. Fang Liang & Yu Zhang & Lin Li & Yexin Yang & Ji-Feng Fei & Yanmei Liu & Wei Qin, 2022. "SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    6. Andrea Rossi & Zacharias Kontarakis & Claudia Gerri & Hendrik Nolte & Soraya Hölper & Marcus Krüger & Didier Y. R. Stainier, 2015. "Genetic compensation induced by deleterious mutations but not gene knockdowns," Nature, Nature, vol. 524(7564), pages 230-233, August.
    7. Antonios Apostolopoulos & Naohiro Kawamoto & Siu Yu A. Chow & Hitomi Tsuiji & Yoshiho Ikeuchi & Yuichi Shichino & Shintaro Iwasaki, 2024. "dCas13-mediated translational repression for accurate gene silencing in mammalian cells," Nature Communications, Nature, vol. 15(1), pages 1-18, December.
    8. Lauren M. Saunders & Sanjay R. Srivatsan & Madeleine Duran & Michael W. Dorrity & Brent Ewing & Tor H. Linbo & Jay Shendure & David W. Raible & Cecilia B. Moens & David Kimelman & Cole Trapnell, 2023. "Embryo-scale reverse genetics at single-cell resolution," Nature, Nature, vol. 623(7988), pages 782-791, November.
    9. Miler T. Lee & Ashley R. Bonneau & Carter M. Takacs & Ariel A. Bazzini & Kate R. DiVito & Elizabeth S. Fleming & Antonio J. Giraldez, 2013. "Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition," Nature, Nature, vol. 503(7476), pages 360-364, November.
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