RNA-targeting CRISPR-Cas optimizations and screenings to understand early development of vertebrates

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. Using our optimized CRISPR-RfxCas13d/CasRx technology, we performed a screening targeting maternally provided mRNAs encoding protein kinases and phosphatases in zebrafish, uncovering Bckdk as a novel post-translational regulator of MZT.

Depletion of bckdk mRNA led to epiboly defects, global ZGA deregulation, reduced H3K27ac levels, and partial impairment of miR-430-dependent maternal RNA degradation. Beyond, phospho-proteomic analysis upon Bckdk depletion revealed a reduced phosphorylation of Phf10, a chromatin remodeling factor also involved in ZGA. Indeed, this lower Phf10 phosphorylation ultimately triggered the developmental phenotype observed in the absence of Bckdk.

Despite our CRISPR-RfxCas13d optimizations in vivo, the activity of CRISPR-RfxCas13d can be further enhanced in vivo, and its application has generated controversy due to the recently described collateral activity in mammalian cells and mouse models.

In our lab, we have continued to improve the CRISPR-RfxCas13d system for an enhanced RNA targeting in vivo using zebrafish embryos as animal model and by different and compatible approaches. Indeed, we demonstrated that:

1. Chemically modified gRNAs increase and maintain mRNA knockdown during early development.
2. Specific nuclear RNA targeting can be more efficient using optimized nuclear localization signals.
3. In vitro-based computational models can predict gRNA efficiency in vivo but with a relatively modest accuracy.

Furthermore, we showed that transient CRISPR-RfxCas13d approaches (ribonucleoprotein complexes or mRNA-gRNA) effectively deplete mRNAs in zebrafish embryos without inducing collateral activity, except when targeting extremely abundant and ectopic RNAs. To circumvent this potential issue, we have implemented alternative RNA-targeting CRISPR-Cas systems such as CRISPR-DjCas13d or CRISPR-Cas7-11 with reduced or absent collateral activity.

Altogether, our findings:

• Demonstrate the potential of CRISPR-RfxCas13d to uncover novel early zebrafish developmental factors, shedding light on the role of Bckdk as post-translational regulator of MZT.
• Contribute to optimize CRISPR-Cas technology for RNA targeting in zebrafish through transient approaches, promoting the development of in vivo CRISPR-Cas knockdown therapies."