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Fig. 5 | BMC Biotechnology

Fig. 5

From: Enhancement of CRISPR-Cas9 induced precise gene editing by targeting histone H2A-K15 ubiquitination

Fig. 5

Knockin of a triple FLAG Tag into endogenous genes in HEK cells. a Schematic drawing of the targeting strategy at the endogenous LMNA, GABPA, CREB1, and AAVS1 loci. HEK cells were transfected with expression vectors for Cas9, sgRNA and a donor vector with tetO elements for introduction of a triple FLAG sequence into the first or last exon of the GABPA (exon 10), CREB1 (exon 9) or LMNA (exon 1) gene and into the AAVS1 site of the PPP1RC12C (first intron) gene, respectively. Three days after transfection, genomic DNA was isolated and the target region was amplified by a two-step PCR reaction using the indicated primers (arrows, green: Illumina adapter). The secondary PCR products were sequenced by amplicon sequencing. b, c, d, e. DSB repair events were quantified by deep sequencing reads for each target gene. The fraction of reads showing HDR (green bars) or Indel events (red bars) is shown on the Y-axis in relation to the total number of reads showing wildtype or gene editing events and was used to calculate the ratio of HDR/NHEJ DSB repair. The table shows the selection of cotransfected plasmids of each sample for the expression of Cas9, sgRNA and BRCA1- and TetR- with Rad18UBD or RNF169UBD fusion proteins. Data are presented as mean values ± SD from two independent experiments. *P < 0.05, **P < 0.01 (HDR) and #P < 0.05, ##P < 0.01 (NHEJ); t-test. Raw data are shown in the Supplementary data file

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