Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

First, we introduced specific chemical modifications into the molecules (guide RNAs) that direct Cas13d, increasing the observed effect (phenotypic penetrance) of modifying embryonic mRNA. We also implemented a protocol to avoid potential toxic effects. Next, we generated a version of the Cas13d protein bound to nuclear localization signals (NLSs), which increases its efficacy against nuclear RNAs. This is crucial for targeting RNAs specifically located in the cell nucleus, such as non-coding RNAs or primary microRNAs. Finally, we compared the ability of computational models developed in mammalian cell cultures to predict the activity of 200 guide RNAs administered in vivo together with Cas proteins.

Furthermore, to expand the toolkit available for future medical and biotechnological applications of RNA-targeting CRISPR-Cas tools, we compared the performance of several Cas proteins. We demonstrated that Cas7-11 and DjCas13d can actively remove extremely abundant and ectopic (GFP) RNAs without causing (or greatly reducing) side effects, with DjCas13d being equally efficient as the original Cas13d.