Figure 2: Allele-specific genome editing in F1 rats by the CRISPR/Cas system. | Nature Communications

Figure 2: Allele-specific genome editing in F1 rats by the CRISPR/Cas system.

From: Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR–Cas platform

Figure 2

(a) Schematic representation of gRNA:Tyrc targeting of the mutant allele (Tyrc) of albino F344 rats and gRNA:TyrC targeting of the wild-type allele (TyrC) of agouti DA rats. (b) Plasmids expressing Cas9 and allele-specific gRNA transfected into F344-derived rat REFs. Cleavage activity by the Surveyor assay was detected with gRNA:Tyrc and with TALENs targeting Tyrc (as a positive control), but not with gRNA:TyrC. M: DNA marker phiX174-HaeIII digest. (c) In DA-derived REFs, cleavage activity was detected with gRNA:TyrC and with TALENs targeting of Tyrc, but not with gRNA:Tyrc. (d) Sequence analysis of the colonies picked from subcloned PCR products from the cultured REFs (b,c). The allele-specific gRNA, and gRNA:Tyrc and gRNA:TyrC showed allele-specific cleavage activity in F344 and DA REFs, respectively, but the TALENs did not act in an allele-specific manner. Data represent the mean±s.d., n=3. *P<0.005 by Student’s t-test. (e) Picture of gRNA:Tyrc-injected (F344 × DA)F1 and (F344 × DA)F1 rats showing the Agouti coat-colour. (f) Some of the gRNA:TyrC-injected F1 rats had albino coloured coats (white arrow) or mosaic coloured coats (grey arrow). (g) Sequence analysis for each of the gRNA-injected F1 hybrid rats. gRNA:Tyrc modified only the F344-allele (Tyrc), while gRNA:TyrC modified only the DA-allele (TyrC) in the F1 hybrid rats. Data represent the mean±s.d., n=3. *P<0.001 by Student’s t-test.

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