Efficient and Precise Integration of Large DNA Sequences Using Precise Interstrand Cross-Linking of Long ssDNA and sgRNA

Homology-directed repair (HDR) allows the precise introduction of functional constructs into the human genome through nonviral gene-editing reagents. However, its application in large DNA sequence gene editing remains limited due to challenges such as low efficiency and the off-target effect. To address these limitations, a new method named AOLP was developed to synthesize chemically modified long single-stranded DNA (lssDNA) as the template donor for Cas9-based gene editing, which has been proven to be more stable than that prepared using the commercial phosphorylation method. We propose a novel strategy involving precise ligation-based interstrand cross-linking between lssDNA and sgRNA using cyanovinylcarbazole nucleoside (CNVK), enhancing the upregulation of the HDR pathway for DSB repair induced by Cas9. The light-activated ligation between Cas9/sgRNA and lssDNA improves the knock-in (KI) efficiency, overcomes the challenges of low KI efficiency, and surpasses the low off-target effect accompanied by the lssDNA donor. Moreover, the interstrand cross-linking of lssDNA and sgRNA can subtly control the ligation sites and the degree of cross-linking of lssDNA and sgRNA to enhance the KI accuracy of HDR. Our approach improves the KI efficiency of lssDNA in K562, HEK293T, and HepG2 cells by 4- to 12-fold relative to conventional lssDNA donors prepared using the phosphorylation method. Furthermore, the KI accuracy of HDR pathway in HEK293T cells is enhanced by >4.7-fold relative to previous commercial lssDNA. Leveraging this approach, we achieved an unprecedented KI rate of approximately 36% for a gene-sized 1.4 kilobase lssDNA insertion in HEK293T cells.