Fig. 3
Impact of small-molecule combinations on targeted nucleotide substitution (TNS) efficiency in iPSCs and hESCs. Shown are TNS efficiencies in CALD1, KATNA1, and SLITRK1 with Cas9n and Cas9, and in HPRT and DNMT1 with Cpf1. Small molecules have an additive effect on TNS efficiency with Cas9n (a) but not with Cas9 (b) in the 409-B2 iCRISPR hiPSC lines. TNS of HPRT and DNMT1 in 409-B2 hiPSCs with recombinant Cpf1 was increased using the CRISPY mix as well (c). Using the CRISPY mix, TNS efficiency was also increased in SC102 A1 hiPSCs and H9 hESCs with plasmid-delivered Cas9n-2A-GFP (GFP-FACS enriched), and in chimpanzee SandraA ciPSCs with recombinant Cpf1 (d). Shown are TNS, TNS + indels, and indels with green, gray, or blue bars, respectively. Error bars show the SD of three technical replicates for a, b, and c, and two technical replicates for d. Concentrations used were 20 µM of NU7026, 0.01 µM of Trichostatin A, 0.5 µM MLN4924, 1 µM NSC 19630, 5 µM NSC 15520, 20 µM AICAR, and 1 µM RS-1. CRISPY mix indicates a small-molecule mix of NU7026, Trichostatin A, MLN4924, and NSC 15520. Statistical significances of TNS efficiency changes was determined using a two-way ANOVA and Tukey’s multiple comparison pooled across the three genes CALD1, KATNA1, and SLITRK1. Genes and treatments were treated as random and fixed effect, respectively. P-values are adjusted for multiple comparison (**P ≤ 0.01, ***P ≤ 0.001). Overall, there was a clear treatment effect (F(12, 24) = 32.954, P ≤ 0.001)
