Nature (London), 2019-04, Vol.568 (7753), p.561-565
2019
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Details
- Autor(en) / Beteiligte
- Titel
- Precise therapeutic gene correction by a simple nuclease-induced double-stranded break
- Ist Teil von
- Nature (London), 2019-04, Vol.568 (7753), p.561-565
- Ort / Verlag
- England: Nature Publishing Group
- Erscheinungsjahr
- 2019
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- 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) and Hermansky-Pudlak syndrome type 1 (HPS1) . 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.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 0028-0836eISSN: 1476-4687DOI: 10.1038/s41586-019-1076-8Titel-ID: cdi_proquest_miscellaneous_2203139491
- Format
- –
- Schlagworte
- Alleles, Bioinformatics, Cell lines, Cells, Cultured, Collapse, Connectin - genetics, CRISPR, CRISPR-Associated Protein 9 - metabolism, CRISPR-Associated Proteins - metabolism, Deoxyribonucleic acid, Disease, DNA, DNA Breaks, Double-Stranded, DNA damage, DNA End-Joining Repair - genetics, DNA repair, Dystrophy, Frameshift mutation, Frameshift Mutation - genetics, Gene mutation, Genetic engineering, Genetic research, Genomes, Genotypes, Hermanski-Pudlak Syndrome - genetics, Hermanski-Pudlak Syndrome - therapy, Hermansky-Pudlak syndrome, Homology, Humans, Lymphocytes B, Methods, Muscular Dystrophies, Limb-Girdle - genetics, Muscular Dystrophies, Limb-Girdle - therapy, Muscular dystrophy, Mutation, Myoblasts - cytology, Myoblasts - metabolism, Myotubes, Nuclease, Nucleases, Nucleotide sequence, Pluripotency, Poly (ADP-Ribose) Polymerase-1 - antagonists & inhibitors, Poly(ADP-ribose) polymerase, Poly(ADP-ribose) Polymerase Inhibitors - pharmacology, Population, Proteins, Repair, Repetitive Sequences, Nucleic Acid - genetics, Ribose, Stem cells