DONOR-FREE GENE CORRECTION BY TARGETED INTERHOMOLOG RECOMBINATION

Abstract

Spontaneous gene correction by interhomolog recombination (IHR) occasionally occurs to ameliorate genetic diseases of blood and skin1-3. Using an engineered endogenous gene as a reporter, we demonstrate that gene correction by IHR is normally infrequent ([≤]0.02%) but is stimulated by DSBs targeted by CRISPR/Cas9 to both homologous chromosomes; reaching frequencies of 0.5%. We further show that depletion of POLQ stimulates IHR frequencies 4-fold, to 2%, and promotes IHR in G2 phase, when recombination between replicated homologs can correct not only compound heterozygous but also autosomal dominant "gain-of-function" mutations, which present a special challenge for gene therapy. The strategies reported here will enable optimization of IHR for gene therapy in a variety of cell types. Advantages include the ability to correct gain-of-function mutations, no need for an exogenous donor, and the potential to limit damage to coding sequence by targeting IHR to introns.