Optimized CRISPR-RfxCas13d system for RNA targeting in zebrafish embryos

CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA knockdown in zebrafish embryos using our in vivo optimized CRISPR-RfxCas13d (CasRx) system. Although the described protocol focuses on mRNA knockdown in zebrafish embryos, it can also be applied to other vertebrates.This work was supported by Ramon y Cajal (RyC-2017-23041), PGC2018-097260-B-I00 grant and MDM-2016-0687 program (Spanish Ministerio de Ciencia, Innovación y Universidades, and European Union), Universidad Pablo de Olavide (UPO) Research and the Springboard programs from UPO and CABD, respectively (M.A.M.-M.). This study was supported by the Stowers Institute for Medical Research. A.A.B. was awarded a Pew Innovation Fund and the US National Institutes of Health (R01 GM136849). This work was performed as part of the research for the obtainment of a G.dS.P degree., Graduate School of the Stowers Institute for Medical Research. The CABD is an institution funded by Pablo de Olavide University, Consejo Superior de Investigaciones Científicas (CSIC), and Junta de Andalucía. L.H.-H. is a recipient of a predoctoral fellowship from Ministerio de Ciencia

CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos

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.This work was supported by Ramon y Cajal program (RyC-2017-23041) and grants PGC2018-097260-B-I00 and MDM-2016-0687 from Spanish Ministerio de Ciencia, Innovación y Universidades and the Springboard program from CABD (M.A.M.-M.) and the Stowers Institute for Medical Research (A.A.B.). M.A.M.-M. was the recipient of the Genome Engineer Innovation 2019 Grant from Synthego. A.A.B. was awarded with Pew Innovation Fund. J.R.M.-M. is supported by BFU2017-86339-P and MDM-2016-0687 grants (Spanish Ministerio de Ciencia, Innovación y Universidades). E.M.-T. and J.A.-N.d.P. are supported by INNOVATE PERÚ grant 168-PNICP-PIAP-2015 and FONDECYT travel grant 043-2019