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.