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Fig. 3 | BMC Biology

Fig. 3

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

Fig. 3

Targeted disruption at CtIP exon-7 yielded cells carrying in-frame variant transcripts. a Schematic for dual reporter knock-in at CtIP using ires-GFP/Tddonor (left), and corresponding FACS data obtained in LO2 cells (right). GFP+ cells are gated to the right, while Td+ cells are gated to the top. Controls without sg-A or sgCtIP are included in right panel. Numbers of single cell clones analyzed and success rates of complete disruption at CtIP are shown. b Genome PCR analysis of CtIPE7ires−/− clones raised from the Td+/GFP single-positive (SP) cells. Primer binding sites are shown in a. c Schematic of CtIP mRNA, sgCtIP target site, and primer binding positions (top); and gel electrophoresis of RT-PCR from selected CtIPE7ires−/− clones (middle), and quantitative analysis by real-time RT-PCR using primers CtIP_F3/R3 (bottom) (Additional file 2: Individual data values for Fig. 3c). d Western blot analysis of selected CtIPE7ires−/− clones. Cells transiently transfected with CtIP cDNA were included. OE:overexpression. e Junction sequences of three aberrant CtIPires transcripts amplified using primers CtIP_F2/R2 in c. The sg-A target sequence from donor is shown in red, and sgCtIP target sequence from genome is in blue. The cleavage sites at the 3rd and 4th nucleotide upstream of PAM in sgCtIP target sequence are indicated with black and blue arrowheads respectively. Other donor sequences are in grey, while other sequences from CtIP genome locus are in black. Two short fragments originated from donor vectors are highlighted in light green and beige. The numbers of base pairs omitted are indicated in brackets. f Schematic showing the modified CtIP alleles harboring reversely integrated ires-Tddonor or ires-GFPdonor (top). Red bars below indicated the positions of sequences detected in the aberrant CtIPires transcripts. Sequences showing cryptic splice sites and the splicing events involved in producing the aberrant CtIPires transcripts (bottom)

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