Scheme of CRISPR/Cas9–gRNA RNP labeling and cell transfection. (A) crRNA and tracrRNA were annealed at room temperature for 10 minutes. The resulting gRNA was labeled with fluorescein- or CX-rhodamine–coupled Label IT Tracker labeling reagent. The fluorescent GADD45B-targeting gRNA was assembled with recombinant Cas9 protein prior to transfection to assemble an active CRISPR/Cas9–gRNA RNP complex targeting human GADD45B. Cells were transfected with TransIT-X2 Transfection Reagent or by using the Amaxa Nucleofector System and were incubated for 24 hours before sorting the CX-rhodamine⁺ or fluorescein⁺ cells using a BD FACSAria II. After sorting, some of the cells were used for a single-cell culture, and the rest were used for DNA isolation or cell-based assays. (B) Virtual gel of an Agilent Bioanalyzer analysis revealing no difference in the size or quality of labeled gRNA compared with unlabeled gRNA. (C) GADD45B was targeted using gRNA (highlighted in red), which inserts a double-strand break at NM_015675.3 exon 1, 31 bp after ATG; NP_056490.2, p.N11.