Homo sapiens Targeted loci cultured. Homo sapiens

We demonstrate efficient repair of microduplications in patient-derived cell lines for two diseases: Limb-Girdle Muscular Dystrophy 2G (LGMD2G)135and Hermansky-Pudlak Syndrome Type 1 (HPS1). Clonal analysis of Streptococcus pyogenesCas9 (SpyCas9)nuclease-treated LGMD2G iPSCs revealed that ~80% contained at least one wild-type allele and that this correction restored TCAPexpression in LGMD2G iPSC-derived myotubes. The Efficient genotypic correction was also observed the uponSpyCas9 treatment of an HPS1 patient-derived B-lymphoblastoid cell line (B-LCL). Inhibition of PARP-1(poly(ADP-ribose) polymerase)suppresses the nuclease-mediated collapse of the microduplication to the wild-type sequence, confirming that precise correction is mediated by the MMEJ(microhomology-mediated end joining)pathway. Analysis of editing by SpyCas9 and Lachnospiraceae bacterium ND2006Cas12a (LbaCas12a)at non-pathogenic microduplications within the genome that range in length from 4 bp to 36 bp indicates that the correct strategy is broadly applicable to a wide range of microduplication lengths and can be initiated by a variety of nucleases. The simplicity, reliability, and efficacy of this MMEJ-based therapeutic strategy should permit the development of nuclease-based gene correction therapies for many50diseases that are associated with microduplications.