Show simple item record

dc.contributor.authorIyer, Sukanya
dc.contributor.authorSuresh, Sneha
dc.contributor.authorGuo, Dongsheng
dc.contributor.authorDaman, Katelyn
dc.contributor.authorChen, Jennifer C. J.
dc.contributor.authorZieger, Marina
dc.contributor.authorLuk, Kevin
dc.contributor.authorRoscoe, Benjamin P.
dc.contributor.authorMueller, Christian
dc.contributor.authorKing, Oliver D.
dc.contributor.authorEmerson, Charles P. Jr.
dc.contributor.authorWolfe, Scot A.
dc.date2022-08-11T08:11:02.000
dc.date.accessioned2022-08-23T17:29:41Z
dc.date.available2022-08-23T17:29:41Z
dc.date.issued2019-04-03
dc.date.submitted2019-06-12
dc.identifier.citation<p>Nature. 2019 Apr;568(7753):561-565. doi: 10.1038/s41586-019-1076-8. Epub 2019 Apr 3. <a href="https://doi.org/10.1038/s41586-019-1076-8">Link to article on publisher's site</a></p>
dc.identifier.issn0028-0836 (Linking)
dc.identifier.doi10.1038/s41586-019-1076-8
dc.identifier.pmid30944467
dc.identifier.urihttp://hdl.handle.net/20.500.14038/50375
dc.description.abstractCurrent 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.
dc.language.isoen_US
dc.relation<p><a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=30944467&dopt=Abstract">Link to Article in PubMed</a></p>
dc.relation.urlhttps://doi.org/10.1038/s41586-019-1076-8
dc.subjectUMCCTS funding
dc.subjectComputational Biology
dc.subjectCongenital, Hereditary, and Neonatal Diseases and Abnormalities
dc.subjectEnzymes and Coenzymes
dc.subjectGenetic Phenomena
dc.subjectGenetics and Genomics
dc.subjectMusculoskeletal Diseases
dc.subjectNervous System Diseases
dc.subjectTherapeutics
dc.subjectTranslational Medical Research
dc.titlePrecise therapeutic gene correction by a simple nuclease-induced double-stranded break
dc.typeJournal Article
dc.source.journaltitleNature
dc.source.volume568
dc.source.issue7753
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/umccts_pubs/202
dc.identifier.contextkey14724588
html.description.abstract<p>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.</p>
dc.identifier.submissionpathumccts_pubs/202
dc.contributor.departmentDepartment of Biochemistry and Molecular Pharmacology
dc.contributor.departmentLi Weibo Institute for Rare Diseases Research
dc.contributor.departmentHorae Gene Therapy Center
dc.contributor.departmentEmerson Lab
dc.contributor.departmentKing Lab
dc.contributor.departmentWellstone Muscular Dystrophy Program
dc.contributor.departmentDepartment of Neurology
dc.contributor.departmentDepartment of Molecular, Cell and Cancer Biology
dc.source.pages561-565


This item appears in the following Collection(s)

Show simple item record