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

Area of Science:

• Molecular Biology
• Gene Editing
• Genetic Diseases

Background:

• Current gene correction methods like CRISPR-Cas9 rely on homology-directed repair, requiring exogenous DNA donors and showing inefficiency in many cell types.
• Microduplications can cause disease-associated frameshift mutations, posing a challenge for precise genetic correction.

Purpose of the Study:

• To develop a more efficient and simpler method for correcting disease-causing microduplication mutations.
• To demonstrate the efficacy of a novel gene correction strategy in patient-derived cell lines.

Main Methods:

• Generating a DNA double-stranded break near the center of microduplications using programmable nucleases (SpCas9 and LbCas12a).
• Utilizing the microhomology-mediated end joining (MMEJ) pathway for precise sequence reversion.
• Testing the strategy in patient-derived cell lines for limb-girdle muscular dystrophy type 2G (LGMD2G) and Hermansky-Pudlak syndrome type 1 (HPS1).

Main Results:

• Efficient reversion of disease-causing frameshift mutations to wild-type sequences in LGMD2G and HPS1 patient cell lines.
• Approximately 80% of treated LGMD2G iPS cells contained at least one wild-type TCAP allele, restoring gene expression.
• Inhibition of PARP-1 suppressed the correction, confirming MMEJ pathway involvement.

Conclusions:

• A novel, nuclease-based strategy efficiently corrects microduplication mutations via the MMEJ pathway.
• This approach is broadly applicable to various microduplication lengths and nucleases, offering a simpler and more reliable gene correction therapy.
• The MMEJ-based strategy holds promise for developing new gene correction therapies for microduplication-associated diseases.