Correction of a Recessive Genetic Defect by CRISPR-Cas9-Mediated Endogenous Repair.

CRISPR-Cas9 technology is a relatively recently developed tool for easy and efficient targeting of DNA. However, its efficiency for the repair of a mutated sequence is low. Moreover, most CRISPR-based gene correction approaches require the use of an exogenous template. Here, we investigated whether we could use the CRISPR-Cas9 system and the autologous repair machinery to correct human recessive genetic disorders having two different mutations in two alleles (compound heterozygotes). We reasoned that by targeting an intronic sequence located between the two mutations, we could generate at least one normal allele via the repair of induced double-strand breaks through either gene conversion or mitotic crossover. In particular, using a simple hypoxanthine-guanine phosphoribosyltransferase (Hprt)-based system, we show we can form a normal and functional Hprt gene. Thus, we give proof of principle that homology-directed recombination can be exploited in compound heterozygote cells to correct a genetic defect without exogenous templates.