Optimizing RNA import for microinjection-free genetic manipulation in C. elegans

Project Summary C. elegans is amenable to easy genetic modifications, as proven by its pioneering and continued use of endogenously expressed fluorescent reporters and other genetically encoded molecular tools. CRISPR/Cas systems for generation of mutants and transgenic lines are widely used in C. elegans. While highly precise, CRISPR/Cas applications are severely limited by the need to microinject nucleic acids to the germline, a significantly time-consuming process that drastically hinders the use of C. elegans for high-throughput biology. On the other hand, C. elegans has the ability to uptake double stranded RNA by feeding, which has led to the generation of dsRNA-expressing bacterial libraries and their use for large-scale reverse genetic screens. In this project, we aim to address this important limitation in C. elegans by developing a strategy that enables microinjection-free CRISPR/Cas applications. We will develop an approach to efficiently deliver RNA by feeding throughout C. elegans tissues for use as single-guide RNA (sgRNA) in CRISPR/Cas systems. Our approach relies in using easily uptaken RNA carriers to facilitate efficient import of sgRNAs through ingestion. We propose to address this challenge by: 1) studying uptake of diverse RNA structures into different C. elegans tissues through the development of an RNA sensor strain, 2) designing RNA structures for optimal uptake using artificial intelligence, and 3) testing the efficacy of modified sgRNA structures in CRISPR/Cas applications. Success of this work will greatly facilitate implementation of CRISPR/Cas approaches and make large-scale high-throughput CRISPR screens possible.