Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases

Abstract

RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the mouse Prkdc gene that encodes an enzyme critical for DNA double-strand break repair resulted in immunodeficiency both in F0 and F1 mice. We propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically engineered model organisms, accelerating functional genomic research.

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

“…We also used RGEN-RFLP to analyse three mutant mice that had been created by injecting a Foxn1-targeting RGEN into one-cell stage embryos 19 . These founder mice carried identical biallelic mutations at the target site (Fig.…”

“…RGENs consist of Cas9, invariable transactivating crRNA (tracrRNA) and target-specific crRNA (or sgRNA that consists of essential portions of tracrRNA and crRNA), and are readily reprogrammed by replacing crRNA (or sgRNA) 7 . We and others have recently used RGENs for genome engineering in bacteria 8 , mammalian cells [9][10][11][12] , animals [13][14][15][16][17][18][19] and plants [20][21][22] .…”

Targeted gene knockout in chickens mediated by TALENs

“…Compared with TALENs, CRISPR-Cas is a fast and cost-effective system for modifying the genomes of various organisms because of its simple, practical use and robust cutting activity (27)(28)(29)(30). CRISPR is an immune system that was discovered in bacteria (31).…”

CRISPR, un système qui permet de corriger ou de modifier l’expression de gènes responsables de maladies héréditaires

“…Plusieurs études initiales ont suggéré que le système CRISPR/Cas9 manquait de spécificité [12,13]. Ce problème a cependant été atténué récemment [14].…”