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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Raw data associated with Extended Data Fig. 10 are reported in Supplementary Table 3. Raw script used for retrieving microduplication data listed in Supplementary Table 3 will be available upon request. Raw Illumina sequencing reads and PacBio data for this study have been deposited in the National Center for Biotechnology Information Short Read Archive under bioproject ID PRJNA517630.
Data analysis used a combination of publicly available software and custom code, as detailed in the Methods. Custom Python (CRESA-lpp.py) and R (indel_background_filtering.R) scripts used in the Illumina data analysis and the shell script (Tcap_pacbio_analysis.sh) used for the analysis of the PacBio data are hosted on GitHub (https://github.com/locusliu/PCR_Amplicon_target_deep_seq). Scripts for the bioinformatic analysis of pathogenic microduplications are hosted at https://rambutan.umassmed.edu/duplications/.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We thank E. Kittler and the UMass Medical School Deep Sequencing Core for sequencing; L. Hayward, L. Qin and D. McKenna-Yasek for coordinating patient enrolment and acquiring patient skin biopsies; Z. Matijasevic for generating LGMD2G iPS cell lines; and the Genome Aggregation Database (gnomAD) and the groups that provided exome and genome variant data to this resource. A full list of contributing groups can be found at http://gnomad.broadinstitute.org/about. This work was supported in part by the National Institutes of Health (R01DK098252, R01HL131471 and R01NS088689 (C.M.); R01AI117839, R01GM115911, R01HL093766 and U01HG007910 (S.A.W.); U54HD0060848 (C.P.E.); a SPARK award through UL1-TR001453) and the Worcester Foundation for Biomedical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Nature thanks Randall Platt and the other anonymous reviewer(s) for their contribution to the peer review of this work.