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. 2020 Sep 1;34(17-18):1227-1238.
doi: 10.1101/gad.339333.120. Epub 2020 Aug 20.

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

Bing Yang  1 , Matthew Schwartz  2 , Katherine McJunkin  1
Affiliations

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

Bing Yang et al. Genes Dev. .

Erratum in

Abstract

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.

Keywords: C. elegans; CRISPR; in vivo screening; miRNA targets; miRNAs; mutational profiling; negative selection screening.

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Figures

Figure 1.
Figure 1.
Mutational profiling coupled with negative selection CRISPR screening for phenotypic mir-35 target sites. (A) Composition of four different simulation pools. Three guide RNAs (targeting sup-26, nhl-2, and selectable marker dpy-10) were diluted 1:4, 1:10, 1:20, and 1:50 by supplementing with gfp guide RNA to maintain a constant total guide RNA concentration. (B) Relationship between dilution factor and indel efficiency (calculated as percent of genotyped alleles in which editing was detected by restriction digest with NciI) (see Supplemental Table S1 for details). (C) Relationship between dilution factor and rate of mutant allele detection per P0 injection. (B,C) Two biological replicates (and mean) are shown. n ≥ 78 per condition. (D) Schematic of mutational profiling of indel positions after CRISPR targeting microRNA-binding sites. (E) Overview of screen workflow. (F) Correlation of indel frequency induced by a given gRNA in two different biological replicates. Spearman's correlation test r and P-value are reported. (G) Distribution of number of alleles detected at each gRNA target site.
Figure 2.
Figure 2.
Mutational profiling identifies candidates with low frequency of seed match-disrupting alleles. (A) Distribution of allele number and percent of seed match-disrupting alleles for each targeted site. (B) Indels detected at the W05B5.1 and egl-1 sites. The top line is the reference sequence of each site. The seed sequence matches are highlighted in yellow, and the expected Cas9 cleavage sites are indicated with arrowheads. Dashes represent deleted bases, whereas gray italic text is insertions.
Figure 3.
Figure 3.
Validation of top candidates confirms egl-1 as a phenotypic target of mir-35. (A) Schematics of 3′UTR and mir-35 mutant genotypes. (B) Brood count and percent embryonic lethality at 25°C of seven seed match-disrupting mutations in the indicated 3′UTRs, compared with wild type. (C) Brood count and percent embryonic lethality at 25°C of seed match-reversing mutations in the indicated 3′UTRs, in the context of wild type or a mutant mir-35 containing compensatory seed mutations. (B,C) Mean and SEM are shown. One-way ANOVA was conducted to determine significance, followed by post hoc pairwise comparisons with correction for multiple testing (Dunnett's test for comparison of each mutant with wild type in B and Sidak's correction for selected pairwise comparisons is shown in C). In C, all single mutants were compared with wild type, and each 3′UTR mutant was compared with the corresponding mutant crossed into the mir-35(rev) background. P-value is indicated only if <0.05. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001.
Figure 4.
Figure 4.
egl-1 is a bona fide phenotypic target of the mir-35 family. (A) Analysis of a reporter transgene that expresses nuclear localized GFP::H2B fusion protein under the control of the constitutive mai-2 promoter and the egl-1 3′UTR. Four embryonic stages are shown. For each embryo, DIC image is shown at the left, and GFP::H2B fluorescence is shown at the right. The mir-35 and egl-1 3′UTR genotype are listed at the left of the images. Scale bar, 10 µm. (B) Quantification of GFP intensity of the corresponding genotypes at the four stages shown. Wild-type (no reporter) samples were treated as negative controls, and their average intensity was subtracted from all experimental samples. Mean and SEM are shown. One-way ANOVA was conducted to determine significance, followed by post hoc pairwise comparisons with Sidak's correction for multiple testing. (****) P < 0.0001.
Figure 5.
Figure 5.
gRNA efficiency correlates with GC content and CrisprScan scores. (A) Comparison of indel efficiencies between GGNGG PAM sites and others. (B) Correlation between the GC content of each guide RNA and its corresponding indel efficiency. (C,D) Correlation of scores predicted by the indicated algorithm for each guide RNA and its observed indel efficiency. Spearman's correlation test r and P-value are reported.
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References

    1. Alvarez-Saavedra E, Horvitz HR. 2010. Many families of C. elegans microRNAs are not essential for development or viability. Curr Biol 20: 367–373. 10.1016/j.cub.2009.12.051 - DOI - PMC - PubMed
    1. Ambros V. 2004. The functions of animal microRNAs. Nature 431: 350–355. 10.1038/nature02871 - DOI - PubMed
    1. Arimbasseri AG, Rijal K, Maraia RJ. 2013. Transcription termination by the eukaryotic RNA polymerase III. Biochim Biophys ActaGene Regul Mech 1829: 318–330. 10.1016/j.bbagrm.2012.10.006 - DOI - PMC - PubMed
    1. Arribere JA, Bell RT, Fu BX, Artiles KL, Hartman PS, Fire AZ. 2014. Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics 198: 837–846. 10.1534/genetics.114.169730 - DOI - PMC - PubMed
    1. Bartel DP. 2009. MicroRNAs: target recognition and regulatory functions. Cell 136: 215–233. 10.1016/j.cell.2009.01.002 - DOI - PMC - PubMed

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