CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos

• Published in: Developmental cell Vol. 54; no. 6; pp. 805 - 817.e7
• Main Authors: Kushawah, Gopal, Hernandez-Huertas, Luis, Abugattas-Nuñez del Prado, Joaquin, Martinez-Morales, Juan R., DeVore, Michelle L., Hassan, Huzaifa, Moreno-Sanchez, Ismael, Tomas-Gallardo, Laura, Diaz-Moscoso, Alejandro, Monges, Dahiana E., Guelfo, Javier R., Theune, William C., Brannan, Emry O., Wang, Wei, Corbin, Timothy J., Moran, Andrea M., Sánchez Alvarado, Alejandro, Málaga-Trillo, Edward, Takacs, Carter M., Bazzini, Ariel A., Moreno-Mateos, Miguel A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28-09-2020
• Subjects: Animals; Cas13d; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR-Cas Systems - genetics; CRISPR-Cas13; early development; embryogenesis; Gene Editing - methods; Gene Expression Regulation, Developmental - genetics; HEK293 Cells; Humans; killifish; knockdown; medaka; MZT; RNA Interference - physiology; RNA targeting; RNA, Messenger - genetics; zebrafish; CRISPR-Cas13; early development; embryogenesis; knockdown; medaka; MZT; killifish; zebrafish; RNA targeting; Cas13d
• ISSN: 1534-5807; 1878-1551
• DOI: 10.1016/j.devcel.2020.07.013

Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.
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•CRISPR-RfxCas13d knocks down maternal and zygotic mRNA in zebrafish embryos•Both RfxCas13d protein and mRNA can be used to recapitulate developmental phenotypes•CRISPR-RfxCas13d is an efficient tool to interrogate embryonic gene function•CRISPR-RfxCas13d is also functional in medaka, killifish, and mouse embryos
The development of mRNA knockdown technologies for use in vertebrate organisms such as zebrafish has been limited. Kushawah et al. establish CRISPR-RfxCas13d as an efficient, specific, cost-effective, and straightforward method for the systematic and tractable study of gene function in vivo during embryogenesis across a range of animal species.