Genome editing is a useful tool in basic andclinical research. Among the several approaches used ingenome editing, the CRISPR–Cas9 system using clusteredregularly interspaced short palindromic repeats (CRISPR)and CRISPR-associated protein 9 (Cas9) along with a guideRNA has been developed recently. The CRISPR/Cas9 systeminduces site-specific double-stranded DNA breaks,which result in DNA repair via non-homologous end joining(NHEJ) or homology-directed repair (HDR). However,HDR effi ciency is lower than that of NHEJ and accordinglyposes a challenge in genome modifi cation studies. Severalchemical compounds including RS-1 have been shown toenhance the HDR knock-in process by two- to six-fold in HEK 293 cells and rabbit embryos. Based on this fi nding,we developed an antibiotic resistance system to screen RS-1chemical derivatives, which may promote effi cient HDR. Inthis study, we report several chemical compounds with highknock-in effi ciency at the ATG5 gene locus, using HeLacell-based assays.