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.

中文翻译：

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在线虫 mir-35-42 家族表型靶标的体内 CRISPR 筛选。

识别 miRNA 靶基因很困难，而划定哪些靶点在生物学上最重要则更加困难。我们设计了一种新策略，通过在秀丽隐杆线虫中通过 CRISPR-Cas9 基因组编辑破坏每个预测的 miRNA 结合位点来测试单个 microRNA 与靶标相互作用的表型影响。我们开发了一种多重负选择筛选方法，其中对编辑的基因座进行深度测序，并根据针对破坏 miRNA 结合的突变的明显选择压力对候选位点进行优先排序。重要的是，我们的筛选是在体内对突变动物进行的，使我们能够询问生物体水平的表型。我们使用这种方法来筛选基本mir-35-42 的表型目标家庭。通过在所有预测靶标中生成 1130 个新的 3'UTR 等位基因，我们将egl-1鉴定为表型靶标，其去抑制通过诱导胚胎致死率和低繁殖力来部分表型复制mir-35-42突变体表型。这些表型可以通过将mir-35重新定位到突变型egl-1 3'UTR的补偿性 CRISPR 突变来挽救。该研究表明，体内全生物体 CRISPR 筛选的应用在加速发现非编码基因组中表型负调控元件方面具有巨大潜力。