Figure 2.

Increasing ssODN concentration reduced KO and improved KI while increasing dgRNA did not affect KO or KI at an optimal Cas9 concentration of 0.4 μM

(A and B) Effect of increased concentrations of Cas9 protein (from 0.2 to 0.5 μM) with Cas9/gRNA/ssODN molar ratios 1/4/4 on KI or B. KO efficiency.

(C and D) Improvement of KI but not D. KO efficiency by increasing Cas9/dgRNA/ssODN molar ratios with 0.4 μM of Cas9.

(E and F) Similar KI and F. KO rates with increased dgRNA concentrations and fixed 1.6 μM of ssODN (1/4 Cas9/ssODN molar ratio).

(G and H) Improved percentage of KI cells and H. reduced percentage of KO cells with increased concentrations of ssODN and fixed 0.4 μM or 1.6 μM dgRNA (1/1 or 1/4 Cas9/dgRNA molar ratio, respectively). Data are represented as individual replicates with mean ± SEM. Replicates from conditions with Cas9/dgRNA/ssODN ratio 1/4/4 with 0.2 μM (N = 3), 0.3 μM (N = 6), 0.4 μM (N = 6) and 0.5 μM (N = 2) of Cas9 (Figures 2A and 2B) and conditions with 0.4 μM of Cas9 with Cas9/dgRNA/ssODN ratios 1/1/1 (N = 3), 1/2/2 (N = 2), 1/3/3 (N = 3) and 1/4/4 (N = 6) (Figures 2C and 2D) were electroporated on different days. Conditions 0.4 μM of Cas9 with Cas9/dgRNA/ssODN ratios 1/2/4 (N = 3), 1/3/4 (N = 3) and 1/4/4 (N = 6) were electroporated on 3, 2 and 6 different days, respectively (Figures 2E and 2F). With 0.4 μM of Cas9, replicates with Cas9/dgRNA/ssODN 1/1/1 (N = 3) and 1/4/4 (N = 6) were electroporated on different days, while ratios 1/1/5 (N = 8), 1/4/2 (N = 3) and 1/4/3 (N = 3) were electroporated on 7, 2 and 2 different days, respectively (Figures 2G and 2H). Nonparametric unpaired Mann-Whitney t-tests have been run between all conditions on each figure. Asterisks highlight the statistical differences between conditions (∗p ≤ 0.05, ∗∗p ≤ 0.01). KI, knockin; KO, knockout; dgRNA, dual guide RNA; ssODN, single-stranded oligonucleotide; hiPSCs, human induced pluripotent stem cells.