Fig. 4

MMEJ-mediated gene knock-in at the hACTB locus in HeLa cells. a Schematic diagram of gene knock-in strategy at the hACTB locus in HeLa cells. Black lines indicate CRISPR target sequences. Black boxes indicate PAM sequences. Black triangles indicate DSB sites. MH, microhomology. Puro, puromycin resistance gene. b Relative frequencies of gene knock-in, fold to mock overexpression. The knock-in frequencies were calculated by FACS analysis, similar to Fig. 3b, except that mCherry was used to normalize the transfection efficiency. Data are expressed as means ± SEM (n = 3). Statistical significance was determined by Student’s t-test. *P < 0.05. c Confocal laser scanning microscopy images of transfected cells. The knocked-in cells showed cytoplasmic localization and peripheral accumulation of mNeonGreen fluorescence. Bars, 30 μm. d, e DNA sequencing analysis of bacterially cloned PCR products of 5’ and 3’ junctions amplified from the cells knocked-in without Exo1 (d) and with Exo1 (e). The intended knocked-in sequence is shown at the top of each set of sequences. Blue bars indicate the microhomologies. Red letters indicate mutated bases including insertions, deletions, and substitutions. Blue letters indicate polymorphisms found on the off-target genomic sites, related to Fig. 5a. Roman numbers shown at the left side of each set of sequences indicate the allele types, related to Fig. 5a and Additional file 1: Figure S15, S16 and Table S1. Statistical significance was determined by chi-square test. *P < 0.05. ** P < 0.01