(A) Experimental schematic of timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) into human cells for genome editing. (B) Chemical inhibitors used to arrest cells at specific phases of cell cycle included lovastatin (Lov), which blocks at early G1 and partially at G2/M phase; mimosine (Mim), aphidicolin (Aph), thymidine (Thy) and hydroxyurea (HU) which arrest cells at the G1-S border prior to onset of DNA replication; and nocodazole (Noc) which causes arrest at G2/M phase. (C) The homology-directed repair (HDR) donor DNA is a 183 nt ssODNA that is complementary to the target sequence (−strand) and contains a 9 nt insertion (HindIII and SphI restriction sequences) at the cut site and a 9 nt deletion downstream of the cut site; these modifications are flanked by 85 nt and 55 nt asymmetrical homology arms at 5′ and 3′ ends, respectively. (D, E) PCR-based screening of cell cycle inhibitors for enhancement of Cas9-triggered total editing (TE) (D) and HDR (E) frequencies in HEK293T cells. For each inhibitor condition (color coded), two doses of Cas9 RNP, 30 and 100 ρmol, were transfected with 100 ρmol of HDR DNA template; control reactions (labeled as C) contained 100 ρmol of Cas9 but no sgRNA. The TE frequency was measured using a T7 endonuclease I assay and analyzed using a formula described in ‘Materials and Methods’. The HDR frequency was determined directly by HindIII digestion, which specifically cleaved the newly integrated HindIII sequence, and calculated as the ratio of DNA product to DNA substrate. The % TE, % HDR and standard deviation (error bars) were calculated from three experiments.