Comparable analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging

CRISPR-mediated endogenous tagging, utilizing the homology-directed repair (HDR) of DNA double-strand breaks (DSBs) with exogenously incorporated donor DNA, is a powerful tool in biological research. Inhibition of the non-homologous end joining (NHEJ) pathway has been proposed as a promising strategy for improving the low efficiency of accurate knock-in via the HDR pathway. However, the influence of alternative DSB repair pathways on gene knock-in remains to be fully explored. In this study, our long-read amplicon sequencing analysis reveals various patterns of imprecise repair in CRISPR/Cas-mediated knock-in, even under conditions where NHEJ is inhibited. Suppression of the microhomology-mediated end joining (MMEJ) or the single strand annealing (SSA) repair mechanisms leads to a reduction in distinct patterns of imprecise repair, thereby elevating the efficiency of accurate knock-in. Furthermore, a novel reporter system shows that the SSA pathway contributes to a specific pattern of imprecise repair, known as asymmetric HDR. Collectively, our study uncovers the involvement of multiple DSB repair pathways in CRISPR/Cas-mediated gene knock-in and proposes alternative approaches to enhance the efficiency of precise gene knock-in.