Efficient repair of human homozygous genetic mutation by CRISPR/Cas9 mediated interlocus gene conversion

Abstract: DNA double-strand breaks (DSBs) induced by gene editing tools are primarily resolved either by non-homologous end joining (NHEJ) or homology-directed repair (HDR) using exogenous synthetic DNA templates. Repaired by error-prone NHEJ may lead to unexpected indels at the targeted site. In the case of most genetic disorders, HDR-mediated precise correction using an exogenous homologous sequence is ideal. However, the therapeutic application of HDR might be especially challenging given the requirement for the codelivery of exogenous DNA templates with toxicity into cells, and the low efficiency of HDR could also limit its clinical application. Here, we used hematopoietic stem cells (HSCs) with genetic mutations to cause β-thalassemia in HBB coding regions and discovered that many cells are actually repaired by CRISPR/Cas9-mediated gene conversion (GC) independent of exogenous synthetic DNA templates. We show that pathogenic mutations in the HBB coding regions of HSCs can be repaired efficiently through CRISPR/GC using the paralog gene HBD as the internal template. Electroporations of Cas9 for ribonucleoprotein with sgRNA into haematopoietic stem and progenitor cells (HSPCs) with a variety of pathogenic gene mutations also resulted in effective conversion of mutations to normal wild-type sequences without exogenous DNA template. Moreover, the edited HSCs can repopulate the haematopoietic system and generate erythroid cells with a greatly reduced propensity for thalassemia after transplantations. Thus, CRISPR/GC, independent of exogenous DNA templates, holds great promise for gene therapy of genetic diseases.