Hijacking-DNA -Repair (HDR)-CRISPR promotes seamless gene editing in human primary cells

The CRISPR-Cas system is a robust platform for genome editing. The introduction of a DNA double strand break (DSB) at precise gene locations can be exploited to achieve targeted gene knockout by harnessing the error-prone non-homologous end-joining (NHEJ) pathway. However, using CRISPR-Cas technology for precise genome editing via homology-directed repair (HDR) remains challenging, with HDR frequencies below the threshold required for clinical translation. Common strategies to increase HDR-mediated DSB repair include the use of chemicals either to inhibit NHEJ or to arrest the cells in those cell cycle phases when HDR is most active. However, the global effects of these drugs pose serious safety concerns if applied in clinically relevant settings. To address this issue, we devised a strategy to recruit HDR-promoting factors or NHEJ-inhibiting proteins to the DSB site. This is achieved via the direct fusion of particular protein-protein interaction domains to the Cas9 nuclease. We generated 16 different Cas9-fusion proteins (referred to as HDR-CRISPR) and extensively investigated their impact on DNA repair pathway choice by using two reporter systems, the traffic light reporter (TLR) and the BFP-to-GFP (B2G) assay. These two assays allowed us to investigate the outcome of DNA repair mediated by a DNA donor supplied either as a plasmid or oligodeoxynucleotides (ODN) respectively. Our results indicate that HDR-CRISPRs enhanced HDR frequency 3-fold over baseline levels. The simultaneous reduction of NHEJ-mediated repair resulted in a 5-fold increase in the HDR:NHEJ ratio when using our best performing HDR-CRSPR. Next, we assessed the capability of HDR-CRISPR to precisely integrate a large GFP expression cassette into the endogenous AAVS1 safe harbor locus of K562 and Jurkat cell lines. Independently of the cell type, the use of HDR-CRISPR resulted in a 2.5-fold increase in targeted integration as compared to samples receiving the unmodified Cas9. The most efficient HDR-CRISPR fusion was then tested for its ability ...