CRISPR-RfxCas13d screenings reveal essential regulators during early development [Extraordinary Nodes Seminar]

The Maternal-to-Zygotic transition (MZT) orchestrates the reprogramming of early vertebrate development, encompassing zygotic genome activation (ZGA) and the clearance of maternally provided RNAs. While some regulators of MZT have been identified, the vast majority of maternal RNAs remain functionally uncharacterized. We have recently implemented CRISPR-RfxCas13d/CasRx technology in animal embryos, and we have demonstrated that it is highly efficient and specific when used through transient approaches such as ribonucleoprotein complexes or mRNA-gRNA. Using our optimized technology, we have performed a set of screenings targeting hundreds of maternally provided mRNAs in zebrafish. As a proof of principle, we have completed a screening focused on mRNAs encoding protein kinases and phosphatases in zebrafish, uncovering Bckdk as a novel post-translational regulator of MZT. Depletion of bckdk mRNA led to early development defects, global ZGA deregulation, reduced H3K27ac levels, and partial impairment of maternal RNA degradation. Beyond, phospho-proteomic analysis upon Bckdk depletion revealed a reduced phosphorylation of Phf10, a chromatin remodeling factor also involved in ZGA. Further, we revealed that this lower Phf10 phosphorylation ultimately triggered the developmental and molecular phenotype observed in the absence of Bckdk. Altogether, our findings i) demonstrate the potential of CRISPR- RfxCas13d to uncover novel early developmental factors shedding light on the role of Bckdk as post-translational regulator of MZT and ii) contribute to optimize CRISPR-Cas technology for RNA targeting in vivo through transient approaches, promoting the development of CRISPR-Cas knockdown therapies.