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.

Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE

“…The pre-implantation embryo stage is generally not favoured for genome editing as it would most likely lead to a mosaic individual and possibly to more unforeseen and unforeseeable detrimental effects. Various studies in different animal models have demonstrated the feasibility of gene-editing in animals at the zygote stage (Yoshimi et al, 2014;Heo et al, 2015;Han et al, 2014, Kang et al, 2014. The potential of the technology to prevent the onset of a genetic disorder in mice was demonstrated by the studies of Wu et al (2013) and Long et al (2014), respectively for cataract and Duchenne muscular dystrophy.…”

Engineered Nucleases Lead to Genome Editing Revolution in Rats

“…We have also generated a targeted KO rat for the coat color gene, tyrosinase ( Tyr ), by microinjecting target-specifi c gRNA and Cas9 mRNA into fertilized eggs (Yoshimi et al 2014 ). Crossing founders demonstrated that the CRISPR/Cas9-mediated mutations were faithfully transmitted to the next generation (Li et al 2013a , b ;Ma et al 2014b ).…”

Don’t edit the human germ line

“…Studies using gene-editing in animals such as rats 5 , cattle 6 , sheep 7 and pigs 8 , indicate that it is possible to delete or disable genes in an embryo -a simpler process than actually correcting DNA sequences -in only some of the cells.…”