HDR template design and KU00060648 treatment. (A) Single-stranded DNA ultramers were used for the KI of HA tag; whereas PCR-generated, double-stranded DNA templates were used for the KI of gfp gene. Red arrows mark Cas9 cleavage sites. GS indicates a glycine-serine linker. The length of homology arms is indicated next to the insert. In the CD96 HDR template, silent mutations were introduced in the sgRNA seed region to avoid Cas9 targeting. In the RAB11A HDR template, a single-base silent mutation was created to disrupt the PAM sequence. The HDR strategies for RAB11A and ACTB were adopted from Roberts et al. (2017) and Roth et al. (2018). The full DNA sequences are listed in Table S5. (B) Treatment of KU00060648 alone, which is a DNA-dependent protein kinase inhibitor and also marketed as a HDR enhancer, produced false GFP signal in a dosage-dependent manner. FSC, forward scatter; PAM, protospacer adjacent motif.