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.
Highlights
The bacterial clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas system has proven to be an efficient gene-targeting tool in various organisms
The CRISPR RNAs, which contain a short stretch of homology to a specific target DNA, act as guides to direct Cas nucleases to introduce double-strand breaks (DSBs) at the targeted DNA sequences
In addition to the rapid creation of synthetic guide RNAs (gRNAs), a significant advantage of the CRISPR/Cas system is its ability to target several genes simultaneously with multiple gRNAs. Another advantage of the CRISPR/Cas system has been described from findings in mice[26,27,33]. These results suggest that co-injected singlestranded oligodeoxynucleotides as donor templates preferentially support the activation of homology-directed repair (HDR) relative to the non-homologous end-joining pathway
Summary
The bacterial CRISPR/Cas system has proven to be an efficient gene-targeting tool in various organisms. Recent progress in the development of genome engineering tools in rats, such as zinc-finger nucleases (ZFNs)[6,7,8] and transcription activator-like effector nucleases (TALENs)[9,10], could provide genetically modified animals for gene annotation, as well as for modelling human genetic disorders These engineered nucleases can recognize long stretches of DNA sequences and introduce DNA double-strand breaks (DSBs), which are generally restored via non-homologous end-joining, a process that introduces small insertions or deletions (indels) at the repair junction, thereby generating knockouts (KOs) at the targeted sequences. We correct the three recessive coat-colour-associated phenotypes that are responsible for the appearance of all ‘albino-white’ laboratory rats using a KI approach that uses ssODN donor templates These results demonstrate the flexible in vivo genome-editing capability of the CRISPR/Cas system and its usability for the creation of genetically engineered animal models of human diseases in rats
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