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

Sukanya Iyer, Sneha Suresh, Dongsheng Guo, Katelyn Daman, Jennifer C. J. Chen, Pengpeng Liu, Marina Zieger, Kevin Luk, Benjamin P. Roscoe, Christian Mueller, Oliver D. King, Charles P. Emerson () and Scot A. Wolfe ()
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Sukanya Iyer: University of Massachusetts Medical School
Sneha Suresh: University of Massachusetts Medical School
Dongsheng Guo: University of Massachusetts Medical School
Katelyn Daman: University of Massachusetts Medical School
Jennifer C. J. Chen: University of Massachusetts Medical School
Pengpeng Liu: University of Massachusetts Medical School
Marina Zieger: University of Massachusetts Medical School
Kevin Luk: University of Massachusetts Medical School
Benjamin P. Roscoe: University of Massachusetts Medical School
Christian Mueller: University of Massachusetts Medical School
Oliver D. King: University of Massachusetts Medical School
Charles P. Emerson: University of Massachusetts Medical School
Scot A. Wolfe: University of Massachusetts Medical School

Nature, 2019, vol. 568, issue 7753, 561-565

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41586-019-1076-8

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