Precise therapeutic gene correction by a simple nuclease-induced double-stranded break

Nature. 2019 Apr;568(7753):561-565. doi: 10.1038/s41586-019-1076-8. Epub 2019 Apr 3.

Abstract

Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication. We demonstrate this in patient-derived cell lines for two diseases: limb-girdle muscular dystrophy type 2G (LGMD2G)1 and Hermansky-Pudlak syndrome type 1 (HPS1)2. Clonal analysis of inducible pluripotent stem (iPS) cells from the LGMD2G cell line, which contains a mutation in TCAP, treated with the Streptococcus pyogenes Cas9 (SpCas9) nuclease revealed that about 80% contained at least one wild-type TCAP allele; this correction also restored TCAP expression in LGMD2G iPS cell-derived myotubes. SpCas9 also efficiently corrected the genotype of an HPS1 patient-derived B-lymphoblastoid cell line. Inhibition of polyADP-ribose polymerase 1 (PARP-1) suppressed the nuclease-mediated collapse of the microduplication to the wild-type sequence, confirming that precise correction is mediated by the microhomology-mediated end joining (MMEJ) pathway. Analysis of editing by SpCas9 and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) at non-pathogenic 4-36-base-pair microduplications within the genome indicates that the correction 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 a variety of diseases that are associated with microduplications.

MeSH terms

  • Alleles
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Associated Proteins / metabolism*
  • Cells, Cultured
  • Connectin / genetics*
  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair / genetics*
  • Frameshift Mutation / genetics
  • Hermanski-Pudlak Syndrome / genetics*
  • Hermanski-Pudlak Syndrome / therapy*
  • Humans
  • Muscular Dystrophies, Limb-Girdle / genetics*
  • Muscular Dystrophies, Limb-Girdle / therapy*
  • Myoblasts / cytology
  • Myoblasts / metabolism
  • Poly (ADP-Ribose) Polymerase-1 / antagonists & inhibitors
  • Poly(ADP-ribose) Polymerase Inhibitors / pharmacology
  • Repetitive Sequences, Nucleic Acid / genetics

Substances

  • CRISPR-Associated Proteins
  • Connectin
  • Poly(ADP-ribose) Polymerase Inhibitors
  • TCAP protein, human
  • PARP1 protein, human
  • Poly (ADP-Ribose) Polymerase-1
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes

Supplementary concepts

  • Limb-girdle muscular dystrophy type 2A