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Fig. 3 | BMC Genomics

Fig. 3

From: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

Fig. 3

Enhancement of MMEJ-mediated gene knock-in by Exo1 overexpression at the FBL locus in HEK293T cells. a Schematic diagram of gene knock-in strategy at the FBL locus in HEK293T cells. Black lines indicate CRISPR target sequences. Black boxes indicate PAM sequences. Black triangles indicate DSB sites. MH, microhomology. Puro, puromycin resistance gene. b Schematic diagram of gene knock-in in HEK293T cells, followed by FACS analysis. c Relative frequencies of gene knock-in, fold to mock overexpression. The knock-in frequencies were calculated by FACS analysis (Additional file 1: Figure S4). Data are expressed as means ± SEM (n = 3). Statistical significance was determined by Student’s t-test. *P < 0.05. d The percentages of mNeonGreen-positive cell areas in mCherry-positive cell areas, calculated by imaging analysis (Additional file 1: Figure S5). Data are expressed as means ± SEM (n = 3). Statistical significance was determined by Student’s t-test. *P < 0.05. e Confocal laser scanning microscopy images of transfected cells. The knocked-in cells showed nucleolar localization of mNeonGreen fluorescence. Bars, 30 μm. f DNA sequencing analysis of bacterially cloned PCR products of 5’ and 3’ junctions amplified from the cells knocked-in with Exo1. The intended knocked-in sequence is shown at the top of each set of sequences. Blue letters indicate the microhomologies. Red letters indicate insertions

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