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

Abstract

The bacterial CRISPR/Cas system has proven to be an efficient gene-targeting tool in various organisms. Here we employ CRISPR/Cas for accurate and efficient genome editing in rats. The synthetic chimeric guide RNAs (gRNAs) discriminate a single-nucleotide polymorphism (SNP) difference in rat embryonic fibroblasts, allowing allele-specific genome editing of the dominant phenotype in (F344 × DA)F1 hybrid embryos. Interestingly, the targeted allele, initially assessed by the allele-specific gRNA, is repaired by an interallelic gene conversion between homologous chromosomes. Using single-stranded oligodeoxynucleotides, we recover three recessive phenotypes: the albino phenotype by SNP exchange; the non-agouti phenotype by integration of a 19-bp DNA fragment; and the hooded phenotype by eliminating a 7,098-bp insertional DNA fragment, evolutionary-derived from an endogenous retrovirus. Successful in vivo application of the CRISPR/Cas system confirms its importance as a genetic engineering tool for creating animal models of human diseases and its potential use in gene therapy.

Figure 1. NHEJ-mediated KO and HDR-mediated KI in Wistar rats using the CRISPR/Cas system.

(a) Schematic representation of the rat tyrosinase (Tyr) gene. The magnified view illustrates the gRNA binding sites (blue) and the PAM sequences (green). Wistar albino rats carry a G896A SNP mutation (orange) in exon 2 of the Tyr gene. (b) Plasmids expressing gRNA and codon-optimized Cas9 were transfected into Wistar-derived Rat-1 fibroblasts. The Surveyor (Cel-I) nuclease assay on exon 2 of Tyr showed targeted cleavage of the digested PCR products (indicated by arrowheads). M: DNA marker phiX174-HaeIII digest. Cas9: Cas9-transfected Rat-1. Cas9 gRNA: Cas9 and gRNA plasmid -transfected Rat-1. (c) Microinjection of gRNA and Cas9 mRNA into fertilized Wistar rat eggs. Sequence analysis of PCR products amplified from the genomic DNA of two-cell embryos showed a wide variety of indel mutations mediated by NHEJ at the targeted Tyr exon 2 (see also Table 1). (d) Co-injection of gRNA, Cas9 mRNA, and ssODN into fertilized Wistar rat eggs. Sequence analysis showed indel mutations at the targeted Tyr exon 2 as well as the precise SNP exchange mediated by HDR that resulted in KI alleles (see also Table 1).

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

(a) Schematic representation of gRNA:Tyr^c targeting of the mutant allele (Tyr^c) of albino F344 rats and gRNA:Tyr^C targeting of the wild-type allele (Tyr^C) 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:Tyr^c and with TALENs targeting Tyr^c (as a positive control), but not with gRNA:Tyr^C. M: DNA marker phiX174-HaeIII digest. (c) In DA-derived REFs, cleavage activity was detected with gRNA:Tyr^C and with TALENs targeting of Tyr^c, but not with gRNA:Tyr^c. (d) Sequence analysis of the colonies picked from subcloned PCR products from the cultured REFs (b,c). The allele-specific gRNA, and gRNA:Tyr^c and gRNA:Tyr^C 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:Tyr^c-injected (F344 × DA)F1 and (F344 × DA)F1 rats showing the Agouti coat-colour. (f) Some of the gRNA:Tyr^C-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:Tyr^c modified only the F344-allele (Tyr^c), while gRNA:Tyr^C modified only the DA-allele (Tyr^C) in the F1 hybrid rats. Data represent the mean±s.d., n=3. *P<0.001 by Student’s t-test.

Figure 3. 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.