A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila

Advances in CRISPR technology have revolutionized molecular biology and greatly facilitated the ability to manipulate nucleic acids. The last decade marked the extensive development of CRISPR/Cas9-based tools that led to new, powerful, and versatile strategies to alter genes or their expression. The recent discovery of a novel type of RNA-targeting endonuclease, Cas13, spawned further studies that showed its great potential as a molecular and therapeutic tool. In this study, we present newly developed Cas13 tools for Drosophila, which work in vivo as well as in cell cultures. To identify Cas13 variants that are optimal for Drosophila, we generated and examined the properties of 40 Cas13 variants in a cell culture system. These efforts resulted in the identification of a single Cas13d variant that outperformed all other tested forms. We also modified Cas13 for specific downstream applications, including validation of RNA-protein interactions, interfering with mitochondrial-encoded transcripts, and targeted RNA editing. Importantly, we present a Cas13/crRNA toolkit that works effectively in vivo and demonstrate that transgenically encoded crRNAs and Cas13 enzymes target endogenous Drosophila transcripts with high efficiency and specificity. Our vector collection allows for both ubiquitous and tissue-specific expression and represents a versatile tool to manipulate gene expression on a post-transcriptional level.