In vivo CRISPR screening for phenotypic targets of the mir-35-42 family in C. elegans

Identifying miRNA target genes is difficult, and delineating which targets are the most biologically important is even more difficult. We devised a novel strategy to test the phenotypic impact of individual microRNA–target interactions by disrupting each predicted miRNA-binding site by CRISPR–Cas9 genome editing in C. elegans. We developed a multiplexed negative selection screening approach in which edited loci are deep sequenced, and candidate sites are prioritized based on apparent selection pressure against mutations that disrupt miRNA binding. Importantly, our screen was conducted in vivo on mutant animals, allowing us to interrogate organism-level phenotypes. We used this approach to screen for phenotypic targets of the essential mir-35-42 family. By generating 1130 novel 3′UTR alleles across all predicted targets, we identified egl-1 as a phenotypic target whose derepression partially phenocopies the mir-35-42 mutant phenotype by inducing embryonic lethality and low fecundity. These phenotypes can be rescued by compensatory CRISPR mutations that retarget mir-35 to the mutant egl-1 3′UTR. This study demonstrates that the application of in vivo whole organismal CRISPR screening has great potential to accelerate the discovery of phenotypic negative regulatory elements in the noncoding genome.