Figure 1.

A traffic light reporter allows for concurrent estimation of gene correction and indel formation in human cells. (A) Schematic overview of TLR, P_CAG: CMV i/e enhancer—chicken β-actin promoter—rabbit globin intron (CAG) promoter; mK02_mut: a variation of the monomeric Kusabira Orange 2 gene with a G67D point mutation in the fluorochrome; PAC: puromycin N-acetyltransferase; HPH: hygromycin-B-phosphotransferase. Graphics in gray shaded area were created with Biorender.com. (B) Detailed view of mK02_mut sequence compared to original mK02. PAM sites for regular SpCas9 CRISPR sgRNAs are depicted in blue (3′ → 5′) and green (5′ → 3′). Amino acids at positions 71 and 73 were modified to allow for efficient ABE targeting. FCYG fluorochrome is framed in black and fluorescence-aborting G67D point mutation is depicted in pink. (C) Example FACS plots depicting 293-TLR cells transfected with Cas9 nuclease and g+4 gRNA or with prime editor and g+1 R14P13 or g+1 R14P13_dP (PAM disrupting) pegRNA. Cells transfected with Cas9 nuclease and an empty sgRNA vector served as unguided control (ugc).