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Resources » Paper

Yang, Bing et al. (2021) International Worm Meeting "In vivo CRISPR screening for biologically important mir-35 targeting sites in C. elegans"

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  • Comments on Yang, Bing et al. (2021) International Worm Meeting "In vivo CRISPR screening for biologically important mir-35 targeting sites in C. elegans" (0)

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    Status:
    Publication type:
    Meeting_abstract
    WormBase ID:
    WBPaper00063561

    Yang, Bing, Schwartz, Matthew, & McJunkin, Katherine (2021). In vivo CRISPR screening for biologically important mir-35 targeting sites in C. elegans presented in International Worm Meeting. Unpublished information; cite only with author permission.

    microRNAs play fundamental roles in maintaining appropriate gene expression levels. They repress multiple target genes through complementarity between the "seed" sequence at position 2-7 of the 5' end of the microRNA and the 3'UTR of the target gene. Identifying miRNA target genes is difficult, and delineating which targets are the most biologically important is even more difficult. Therefore, 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. 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.

    Affiliations:
    - 1National Institute of Diabetes and Digestive and Kidney Diseases Intramural Research Program, National Institutes of Health, Bethesda, Maryland 20815, USA
    - Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA


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