Fig. 3.

Simultaneous generation of multicolored cell collections. a Schematic of triple gene knock-in with the enhanced PITCh system. Three independent loci were targeted by a single-donor vector containing green, red, and blue fluorescent protein genes flanked by specific microhomologous arms for each gene locus. Triple gene knock-in was achieved by co-transfection of the integrated donor vector, all-in-one CRISPR vector, and MS2-CtIP vector. U6, human U6 promoter; CBh, chicken beta-actin short promoter; CMV, cytomegalovirus promoter. b A merged low-power image of heterogeneous cell populations transfected with triple knock-in vectors illustrated in Fig. 1a, targeting FBL, CANX, and ATP5B genes. Differential interference contrast and green, red, and blue fluorescence images were merged. A magnified view of the region boxed in black is displayed in the bottom-left corner. Bar, 100 μm. c Merged high-power images of triple knock-in cells transfected with knock-in vectors with various combinations of target genes with fluorescent protein genes. Green, red, and blue fluorescence images were merged. Bar, 30 μm. d Examples of the fluorescence images of various single, double, and triple knock-in cell clones simultaneously generated by a single transfection and single-cell cloning for each combination. Bar, 30 μm. e Numbers and percentages of single, double, and triple gene knock-in clones in the two types of triple gene knock-in. f Sanger sequencing of knock-in junctions of triple knock-in clones. The intended knock-in sequences are shown at the top of each sequence. Green, red, and blue letters indicate the coding sequences of mNeonGreen, mKate2, and TagBFP, respectively. Dashes indicate deletions. Underlines indicate microhomologies