Transfection- and genome-editing efficiency in different cell types using CX-rhodamine–labeled CRISPR/Cas9–gRNA RNP targeting GADD45B. (A) HEK293FT cells were transfected with CX-rhodamine–labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP. Fluorescence signal could be detected for up to 72 hours posttransfection. Representative images of 3 experiments are shown. (B) HEK293FT cells, Jurkat cells, human iPSCs, and CD34⁺ cells were transfected with labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP. At 24 hours posttransfection, cells were harvested and measured for transfection efficiency using a BD FACSCanto II flow cytometer. Representative line graphs of 3 independent experiments are shown. (C) HEK293FT cells, Jurkat cells, human iPSCs, and CD34⁺ HSPCs were transfected with unlabeled or labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP and analyzed for gene-modification efficiency using a TIDE assay. (D) Jurkat cells, human iPSCs, and CD34⁺ HSPCs were transfected with CX-rhodamine–labeled GADD45B-targeting CRISPR/Cas9–gRNA RNP and sorted 24 hours posttransfection using a flow cytometer. Genomic DNA was isolated 48 hours posttransfection from the total population of transfected cells and from sorted CX-rhodamine⁺ or fluorescein⁺ cells. TIDE assay analysis showed significantly higher gene modification efficiency in CX-rhodamine⁺ cells. Data in panels C and D are mean ± standard deviations derived from 3 (HEK293FT cells, Jurkat cells, CD34⁺ HSPCs) or 4 (iPSCs) independent experiments. *P ≤ .05, **P ≤ .01, Student t test. ns, not significant.