Fig. 3

Post-cleavage residency of LbCas12a at the PPEs inhibits recruitment of Ku80 to the ends. a Schematic depicting end-binding competition between HA-tagged LbCas12a and c-NHEJ core factors at LbCas12a-induced DSBs. Ku70/Ku80 is expected to bind the PDE of LbCas12a-induced DSBs, while LbCas12a-gRNA remains bound at the PPE. Enrichment of HA-tagged LbCas12a and Ku80 at the PPE and PDE of a DSB induced by LbCas12a-gRNA-W in the SCR-RFP reporter (b) and by LbCas12a-gRNA-C in the NHEJ reporter (c). Mouse ES cells containing either the SCR-RFP reporter or the NHEJ reporter were transfected with expression plasmids for LbCas12a and the corresponding gRNA. Inset in b and c indicates T7E1 detection of on-target cleavage by LbCas12a-gRNA-W and LbCas12a-gRNA-C at 48 h post-transfection, along with gU6 as a negative gRNA control. In these insets, uncut and cut PCR bands are indicated, along with M for DNA marker, and the indel ratios were calculated according to the band intensities with ImageJ software. ChIP analysis was performed at 24 h post-transfection using anti-IgG as a background control (left), anti-Ku80 antibody for Ku80 enrichment (middle), and anti-HA antibody for HA-LbCas12 enrichment (right). Fold enrichment of HA-LbCas12a and Ku80 was assessed by real-time qPCR amplification using primer pairs located at varying distances from the LbCas12a cleavage site, as indicated by the orange and blue arrows. The fold enrichment relative to the negative IgG control was normalized to 1. Columns with error bars represent the mean ± S.D. of three independent experiments, with statistical significance detected by Student’s two-tailed paired t-test in b and c. *: P < 0.05; **: P < 0.01