Enhancing Precision Genome Editing by Improving Homology-Directed Repair using Cas9-Exonuclease Fusion

Abstract

Since the discovery of the DNA’s structure nearly seventy years ago, the diagnosis of genetic disorderss has advanced rapidly, thanks to the declining financial burden of genome sequencing, the enhancement of computational methods for analyzing and comparing human genome sequences, and the broader use of large-scale genomic screening. As a result, over 3,000 mutated genes have been identified as associated with disease-related phenotypes, with the discovery of new genetic diseases continuing at a rapid pace. Such advancements have created a growing gap between diagnostic advancements and therapeutic developments, underscoring the importance of precision genome-editing technologies in bridging that gap. Here, the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR- associated proteins) has emerged as a revolutionary tool, offering unparalleled precision and efficiency. Yet relatively low efficiency of Homology-directed repair (HDR) limits precision genome editing. DNA end resection plays a critical role in guiding the choice of repair pathways away from the dominant competing pathway, non-homologous end joining (NHEJ), by creating 3' single-stranded DNA (ssDNA), which can search for and invade a homologous strand. Given the temporal and structural importance of 3' end formation in HDR, we hypothesized that longer, faster-mediated 3' ends could function as more efficient strand invaders. With this objective, we fused human EXO1 (hEXO1) to SaCas9 protein, to force 5’ end resection right after DNA cleavage induction. We demonstrate that our hEXO1-SaCas9 fusion protein increases the frequency of HDR pathway usage by 2-fold and enhances large gene knock-in efficiency by 1.5 to 2-fold compared to SaCas9. hEXO1-SaCas9 also significantly reduces deletion products at DNA double-strand break sites (DSBs), leading to a decrease in the overall mutation rate compared to SaCas9. Finally, as the ultimate goal in gene editing, we sought to determine whether our fusion protein could perform more effectively in gene editing applications, and we observed a 24% improvement in gene correction in patient- derived cells.