419 Efficient CRISPR/Cas9-mediated knockout of Col7a1 in NOD/SCID IL2Rgamma-null mice by pro-nuclear injection

Abstract

Current immunocompetent Col7A1-null mouse models of recessive dystrophic epidermolysis bullosa (RDEB) are not amenable to studies involving the transplantation of human cells. To address this limitation, we used pro-nuclear injection and CRISPR/Cas9 technology to disrupt Col7a1 in immunodeficient NOD/SCID IL2Rg-null (NSG) embryos; a strain permissive to adoptive transfer of human cells. Tandem guide RNAs (gRNA) targeting exon two of Col7a1 were co-delivered with Cas9 into single-cell embryos via pronuclear injection. Utilizing low and high dose, we obtained pups exhibiting phenotypic manifestations of RDEB at rates of 34% (n = 35) and 83% (n = 79) respectively. Complete absence of type VII collagen in skin and mucosa of blistered pups was confirmed by immunofluorescence microscopy. There was no evidence of mosaicism, indicating that molecular disruption occurred at the single-cell stage. We observed a range of CRISPR/Cas9 induced indels that included mutation at each site individually and concurrently, as well as deletions spanning both target sites. Non-blistered pups with single allele frameshift disruptions were used to establish breeding colonies. The short lifespan of Col7A1-null mice necessitates carefully timed mating, and the number of homozygous-null animals is limited by Mendelian genetics. Using pro-nuclear injection and CRISPR/Cas9 we routinely obtained large numbers (>30) of RDEB-NSG pups born within narrow time windows (<12 hours), thereby facilitating complex human cellular transplant experiments. The ease with which CRISPR/Cas9 can be directed to disrupt discreet genomic loci with high fidelity will facilitate the rapid production of disease model mice on the NSG background without the need for time-consuming and expensive backcrossing, accelerating pre-clinical trials testing the safety and efficacy of cellular therapies.