Figure 3.

P53DD modRNA increased Cas9 modRNA-mediated gene KO in hPSCs

(A) Schematic of optimal transfection protocol with the addition of p53DD modRNA.

(B) Aggregated gene KO efficiencies across multiple replicates and batches in H1 OCT4-GFP cells, comparing results between transient plasmid DNA transfection and modRNA-based delivery with or without p53DD as well as RNP lipofection (plasmid: n = 9; modRNA: n = 20; plasmid + p53DD: n = 6; modRNA + p53DD: n = 13; RNP: n = 3).

(C) H9 cells cultured on iMatrix-511 in mTeSR1 were transiently transfected with either the plasmid DNA with or without p53DD plasmid, modRNA cocktail with or without p53DD modRNA, or Cas9 RNP. On day 5, cells were collected, and CD90 expression was analyzed via flow cytometry.

(D) Aggregated CD90 KO efficiencies across multiple replicates and batches in H9 cells, comparing results between transient plasmid DNA transfection and modRNA-based delivery without or with p53DD as well as RNP lipofection (plasmid: n = 6; modRNA: n = 8; plasmid + p53DD: n = 6; modRNA + p53DD: n = 14; RNP: n = 3; one-way ANOVA with post-hoc Tukey’s test).

(E) Genotype of CD90KO H9 cells generated using CRISPR modRNA cocktail with p53DD modRNA (n = 8).

(F) G-banded karyotype analysis of CD90 KO H9 cells generated using modRNA cocktail with p53DD.

(G) IMR90C4 cells cultured on iMatrix-511 in mTeSR1 were transfected with Cas9Puro modRNA, CD90 sgRNA, and p53DD modRNA. On day 5, cells were collected, and CD90 expression was analyzed via flow cytometry (H9: n = 14; IMR90C4: n = 12).

(H and I) H1 OCT4-GFP cells were cultured on iMatrix-511 in mTeSR1 using a 12-well plate and transfected with 1,200 ng Cas9Puro modRNA, 200 ng CD90_1 sgRNA, 200 ng GFP sgRNA, and 200 ng p53DD modRNA. On day 5, cells were collected, and GFP/CD90 expression was analyzed via flow cytometry (n = 3).

(H) Representative flow cytometry plot from day 5.

(I) Quantification of flow cytometry results from day 5 cells.

See also Figures S3 and S4.