Interrogation of Functional miRNA–Target Interactions by CRISPR/Cas9 Genome Engineering
Post-transcriptional silencing by microRNAs (miRNAs) is a critical constituent of eukaryotic gene regulation. miRNAs are short (~22nt) noncoding RNAs capable of specifically targeting the miRNA-induced-silencing-complex (miRISC) to transcripts bearing a complementary miRNA response element (MRE). Although recent methodological advances have greatly improved our understanding of miRNA biogenesis and the mechanisms by which miRNAs repress their cognate targets, exploring the physiological relevance of direct miRNA–target interactions in vivo has remained an outstanding challenge. Here we describe the experimental protocol underlying a novel approach, which allows direct interrogation of specific miRNA–MRE interactions by CRISPR/Cas9-mediated genome engineering. In this instance, the CRISPR/Cas9 system is first used to catalyze homology-directed replacement of candidate MREs with molecular barcodes at endogenous loci. Subsequently, the effect of MRE mutation on transcript abundance (i.e., MRE activity) can be rapidly evaluated by routine quantitative PCR. This strategy enables functional investigation of a putative miRNA–target pair in a pool of transiently transfected cells, obviating the need for generation of clonal cell lines or transgenic animals. This protocol can be implemented in any cell line in less than 2 weeks, and can readily be scaled up for multiplex studies. To facilitate the conceptual workflow underlying this strategy, we also describe a genome-wide resource for automated design and computational evaluation of CRISPR/Cas9 guide RNAs targeting all predicted MREs in various species (miR-CRISPR).
Key wordsmicroRNAs miRNA miRNA response elements (MRE) Genome engineering CRISPR/Cas9
We thank Andrew Basset for critical comments on the manuscript and the Fulga lab for feedback and advice. Y.S.M is supported by the Clarendon Scholarship; WIMM Prize Fellowship; Christopher Welch Scholarship. Q.W is supported by the Medical Research Council (WIMM Strategic Award, MRC #G0902418 to T.A.F.). T.A.F. is supported by the Medical Research Council (WIMM Strategic Award, MRC #G0902418); Biotechnology and Biological Sciences Research Council (Project Grants #BB/L010275/1 and #BB/N006550/1); Welcome Trust ISSF Award (#105605/Z/14/Z).
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