Journal: Nature Communications
Article Title: Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR-Cas platform
Figure Lengend Snippet: Recovery of three distinct coat-colour mutations by CRISPR/Cas. ( a ) Schematic illustration of three coat-colour mutations in rats. albino ( Tyr c ): SNP missense mutation in the Tyr gene. non-agouti ( Asip a ): 19-bp deletion in exon 2 of the Agouti signalling protein ( Asip ) gene. hooded ( Kit h ): integration of an 7,098-bp endogenous retrovirus (ERV) element within the first intron of the Kit gene. ( b ) Coat-colour phenotypes ( C, a, h ) recovered from albino by injecting gRNA: Tyr c , Cas9 mRNA, and ssODN of the Tyr C allele into F344 rat embryos ( c, a, h ). ( c ) Sequence analysis of the targeted Tyr exon 2 in the injected F344 rats. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise HDR-mediated SNP exchange of Tyr C . ( d ) Recovery of the non-agouti phenotype by injecting gRNA: Asip a , Cas9 mRNA, and ssODN of the Asip A allele into F344 rat embryos. Cas9 and gRNA with ssODN mediated the introduction of several indel mutations, and the precise short DNA fragment integration of the Asip A gene . ( e ) Recovery of the hooded mutation by injecting gRNA: Kit h -1 , gRNA: Kit h -2 , and ssODN of the Kit H allele into F344 rat embryos. Cas9 and two gRNAs with ssODN mediated the introduction of indel mutations at the targeted Kit h -1 locus, and the precise large deletion between the two cutting edges of Kit H . ( f ) PCR analysis of the injected F344 rats using primers designed against each outer side of the two LTR sequences. M: DNA marker phiX174-HaeIII digest.
Article Snippet: To prepare Cas9 mRNA and gRNA, T7-promoter Cas9 and gRNA expression plasmids were linearized with Xho I and Hind III, respectively, and extracted with NucleoSpin Gel and PCR Clean-up kits (Macherey-Nagel, Düren, Germany).
Techniques: CRISPR, Mutagenesis, Sequencing, Injection, Polymerase Chain Reaction, Marker