Ms. The Chinese University of Hong Kong (CUHK), Hong Kong
Abstract: CRISPR-Cas9-mediated insertion of exogenous sequences into a targeted genomic locus showed promising potential to provide novel gene therapies to treat a wide range of inherited disorders. However, low integration rates and diverse editing outcomes remain the major hurdles for clinical translation. In this study, haemophilia B was employed as a disease model to assess the therapeutic potential of in vivo targeted insertion strategies. We performed a multi-parameter knock-in analysis, to identify efficient and high-fidelity gene knock-in strategy for advancing gene insertion-based therapy. A systematic and quantitative framework using Illumina RNA-seq and long-read sequencing was employed to analyze diverse knock-in outcomes. In this study, we performed a comparative analysis of multiple parameters involved in knock-in process. The results revealed that break configurations, choice of DNA repair pathways and donor template play an important role in the targeted insertion. Cas9-induced DSBs are necessary for efficient knock-in. Nickase-induced SSB (single-stranded break) and no cutting groups showed low or even undetectable insertional efficiency. Surprisingly, we found that leveraging distinct DNA repair pathways yields comparable insertional efficiency in mouse liver. Surprisingly, we found that only 20bp-microhomology arms in MMEJ (microhomology-mediated end joining) donor could support insertion as efficient as the widely demonstrated HDR (homology-directed recombination) donor and NHEJ (non-homologous end joining) donor. The chimeric donor containing only one homology arm showed suboptimal performance. Besides, we observed that ssAAV vector outperformed the scAAV as a donor template for targeted insertion over scAAV, no matter combining with NHEJ donor or MMEJ donor. In conclusion, a comparative analysis of parameters highlights the role of break configurations, DNA repair pathways and donor template in the targeted insertion. Future research will leverage long-range sequencing to analyze diverse insertional outcomes influenced by these parameters.
