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Targeted mutagenesis in a human-parasitic nematode

Fig 4

CRISPR-mediated mutagenesis of Ss-unc-22 results in putative deletion of the target locus.

(A) Representative gel of wild-type iL3s (top) or unc F1 iL3s from RNP injections at site #3 (bottom). Genomic DNA from each iL3 was split into two reactions: ctrl. = control reaction amplifying 416 bp of the first exon of the Ss-act-2 gene to confirm the presence of genomic DNA; u22 = reaction amplifying 660 bp around site #3. Size markers = 1.5 kb, 1 kb, and 500 bp from top to bottom. (B) The Ss-unc-22 region is significantly depleted in unc F1 iL3s. Left: relative quantity analysis of PCR products. All control bands and all u22 bands were quantified relative to their respective reference bands, denoted by asterisks in A. Values >1 indicate more PCR product than the reference while values <1 indicate less product. ***P<0.001, two-way ANOVA with Sidak’s post-test. Medians and interquartile ranges shown. Right: relative quantity of the Ss-unc-22 site #3 target region for each unc F1 iL3 tested, and inferred genotypes. nd = PCR product not detected. (C) Whole-genome sequencing coverage plots for populations of Ss-unc-22-targeted F1 iL3s or wild-type iL3s. A 4-kb window centered on the predicted cut site is shown [24,47]. Black lines = average coverage depth by position (reads per base); red lines = average genome-wide coverage; blue lines = average coverage for the Ss-unc-22 gene. Coverage around Ss-unc-22 site #3 is significantly depleted in both Ss-unc-22 libraries relative to the Ss-unc-22 gene average (P<0.05; see Methods). No depletion is observed in the wild-type library (P>0.05; see Methods). Gray shaded regions represent stretches of continuous significant coverage depletion around the cut site (Ss-unc-22 library A = 510 bp, Ss-unc-22 library B = 725 bp).

Fig 4