Enhanced RNA-targeting CRISPR-Cas technology in zebrafish V

Transient elimination of RNAs by CRISPR-Cas13 systems has been widely used in basic and applied sciences over the last years. However, the efficiency of the system can be enhanced in vivo, and its application has generated controversy due to the recently described collateral activity in mammalian cells and mouse models. Here, we have optimized the CRISPR-RfxCas13d (CasRx) system for an optimized RNA targeting in vivo in zebrafish embryos, by different and compatible approaches. These strategies include the use of chemically modified guide RNAs to increase and sustain mRNA knockdown, an improved nuclear targeting, and the evaluation of ex vivo computational models for predicting gRNA efficiency in vivo. Furthermore, our study demonstrated that transient CRISPR-RfxCas13d approaches can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except for targeting extremely abundant RNAs. For that, we have implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity in zebrafish embryos. Altogether, these findings contribute to optimize CRISPR-Cas technology for RNA targeting in zebrafish through transient approaches and assist the progress of potential implications for knockdown therapies in vivo. To investigate the toxicity effect of IVTed gRNAs compared with chemically synthesized