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Targeted mutagenesis in a human-parasitic nematode

Fig 5

CRISPR-mediated homology-directed repair of Ss-unc-22.

(A) Strategy for HDR at Ss-unc-22 target site #2. unc F1 iL3s that displayed both the nicotine-twitching phenotype and red fluorescence were selected as candidates for HDR and were genotyped using the primer sets indicated. 5’ and 3’ integration primer pairs amplify only following successful integration of Ss-act-2::mRFPmars into site #2. HA = homology arm. (B) Representative DIC + epifluorescence overlays of unc F1 iL3s expressing Ss-act-2::mRFPmars. Top, iL3 expressing mRFPmars (sparse expression indicated by the arrow) from an extrachromosomal array. Bottom, iL3 expressing mRFPmars following HDR, showing near-uniform mRFPmars expression in the body wall. For both images, anterior is to the left. Scale bar = 50 μm. (C) Representative genotypes of a wild-type iL3 and unc F1 iL3s expressing mRFPmars. Genomic DNA from individual iL3s was split into four reactions: ctrl. = control reaction amplifying 416 bp of the first exon of the Ss-act-2 gene to confirm the presence of genomic DNA; wt = reaction for the wild-type locus of site #2 where primer R1 overlaps the predicted CRISPR cut site; 5’ = reaction for insertion of the 5’ border of the integrated cassette; 3’ = reaction for insertion of the 3’ border of the integrated cassette. For genotypes: array = red unc F1 iL3s that showed no evidence of integration; integrated = red unc F1 iL3s with successful HDR. Some integrated iL3s had putative homozygous disruptions of Ss-unc-22 site #2 (e.g. iL3s #4 and #7, which lacked the wt band). Asterisks indicate genotypes for iL3s shown in B. Size markers = 2 kb, 1.5 kb, 1 kb, and 500 bp from top to bottom.

Fig 5