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Fig. 2 | BMC Biology

Fig. 2

From: Homology-independent multiallelic disruption via CRISPR/Cas9-based knock-in yields distinct functional outcomes in human cells

Fig. 2

Complete disruption of ULK1 and FAT10 genes through simultaneous knock-in of dual reporters. a Schematic for dual reporter knock-in at ULK1 and FAT10 using ires-GFP/Tddonor (left), and corresponding FACS data obtained in LO2 cells (right). sgFAT10-i and sgFAT10-ii represent two different sgRNAs targeting FAT10 exon-2. GFP+ cells are gated to the right, while Td+ cells are gated to the top. sg-G represents any sgRNA targeting ULK1 or FAT10 genes. Controls without sg-A or sg-G are shown. b Genome PCR analysis of pooled Td+/GFP+ and Td+/GFP cells collected from the targeting at ULK1. Primer binding sites are shown in a. c Western blot analysis of sorted Td+ /GFP+ and Td+ /GFP− cell populations targeted for ULK1. Numbers shown are ULK1 protein normalized to β-actin.  d Numbers of single cell clones analyzed and success rates of complete disruption from the targeting at ULK1 or FAT10. e Schematic of ULK1 mRNA and primer binding sites (top), and corresponding RT-PCR results (bottom). GAPDH was included as control. f Western blot analysis of ULK1−/− clones, using antibodies against ULK1 and β-Actin. g Mitochondria staining with MytoTracker Red in wild-type LO2 cells and ULK1−/− clones. Shown are images taken after treatment with Oligomycin (10 μM) and Antimycin A (1 μM) for 24 h. Untreated cells were included as controls. Scale bars = 20 μm. h Schematic of FAT10 mRNA and primer binding sites (top), and RT-PCR analysis (bottom). Cell samples were treated with TNFα and INFγ. GAPDH was included as control. i Western blot analysis of FAT10−/− clones, using antibodies against FAT10, GFP and β-Actin. Cells were treated with TNFα and INFγ. Untreated cells were included as control. j Cell viability measured using MTT assays. Shown are percentages of growth rate after treatment with TNFα and INFγ, compared to untreated cells. The measurements were done in wild-type LO2 cells and FAT10−/− clones. k Reversal of the increased cell viability in FAT10−/− clone F-13 after overexpression of FAT10 cDNA. Cell viability measured using MTT assays. Each column represents the mean ± s.d. of the six replicates in j, or of the four replicates in k (Additional file 2: Individual data values for Fig. 2j and k). *p ≤ 0.05

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