ENGINEERING DNA REPAIR PATHWAYS TO IMPROVE THERAPEUTIC GENOME EDITING ACCURACY

Abstract

Background: Precision health care has been revolutionized by CRISPR-Cas9 technology for therapeutic genome editing, but inaccurate DNA repair and non-target mutations are major obstacles for clinical application. However, nonhomologous end joining (NHEJ) frequently results in undesired insertions and deletions, lowering editing fidelity as well as genomic stability.

Objective: To enhance the accuracy of therapeutic genome editing by engineering DNA repair pathways to enhance Homologous Recombination Directed Repair (HDR) and inhibit error-prone repair pathways.

Methodology: CRISPR-Cas9 systems were used to edit human cell lines and DNA repair process modulation strategies by RAD51 expression, whereas DNA-PK inhibition and CtIP activation. were implemented. Editing accuracy, HDR efficiency, as well as off-target the incidence of mutations were evaluated through next-generation sequencing protocols, fluorescence reporter evaluations, and molecular validation methods.

Results: The engineered repair system showed a 42% increase in HDR-mediated precise genetic correction and a decrease of 35% in off-target mutations in comparison to conventional CRISPR editing. Further optimization of the repair pathway improved cell viability from 82% to 91%.

Conclusion: Engineering regarding DNA repair pathways greatly improves the accuracy of therapeutic genome editing, genomic stability and clinical safety, which may provide a viable approach for future precision health care applications.