Stepwise Evaluation of Plasmid‐ and Adeno‐Associated Virus‐Based Knock‐In Using A Triple‐Reporter Platform

Homology-directed repair (HDR)-mediated knock-in efficiency is a composite of double-strand break (DSB) induction and repair pathway selection during targeted insertion. Thus, optimization of HDR-mediated knock-in presents significant challenges. To address this, we employed a mouse embryonic stem cell-based triple-reporter platform to examine how donor design and experimental parameters are associated with DSB induction and the balance between HDR-mediated knock-in and end-joining-mediated targeted insertion (EJ-TI). Our analysis reveals that donor design directly impacts DSB induction: co-electroporation of donor plasmids with Cas9/gRNA reduced cleavage efficiency, and Homology-Independent Targeted Integration (HITI) sequences further reduced this efficiency, thereby biasing overall knock-in efficiency. When normalized to DSB levels, HITI increased total targeted insertion yield (knock-in + EJ-TI) with shorter arms and favored HDR-mediated knock-in over EJ-TI with longer arms. Conversely, under adeno-associated virus (AAV) donor conditions, HITI showed donor-format dependency. It increased knock-in and decreased EJ-TI only with a self-complementary AAV donor, but had little effect with a single-stranded AAV donor. These results highlight the importance of separately evaluating DSB induction, total targeted insertion yield, and the balance between HDR-mediated knock-in and EJ-TI using a triple-reporter platform when optimizing knock-in strategies.