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Fig. 5 | BMC Biology

Fig. 5

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

Fig. 5

Targeted disruption at CtIP 5′-UTR also produced cells carrying splice variant transcripts. a Schematic for simultaneous knock-in of 5’GFPdonor and 5′Tddonor at CtIP 5′-UTR (left); and FACS plot 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 sg5′CtIP are shown. b Western blot analysis of pooled Td+/GFP+ double-positive (DP) and Td+/GFP single-positive (SP) cells collected from a. Cells transiently transfected with CtIP cDNA were included. Numbers shown are CtIP protein levels normalized to β-actin. OE, overexpression. c RT-PCR analysis of selected CtIP5’UTR −/− clones. Shown are schematic of CtIP mRNA, sg5′CtIP target site, and primer binding positions (top), gel electrophoresis of RT-PCR products (bottom left), and quantitative RT-PCR analysis using primers CtIP_F3/R3 (bottom right) (Additional file 2: Individual data values for Fig. 5c). d Western blot analysis of the selected CtIP5UTR −/− clones. Cells transiently transfected with CtIP cDNA were included as positive control. OE, overexpression. e Sequences of the RT-PCR products amplified from aberrant CtIP5UTR transcripts with primers CtIP_F5/R5* in c. Shown are junction sequences of two aberrant CtIP5UTR transcripts. The sg-A target sequence is in red, and sg5′CtIP target sequence at CtIP 5′-UTR is in blue. The cleavage sites at the 3rd and 4th nucleotide at upstream of PAM in sg-A target sequence are indicated with black and red arrowheads respectively. Other donor sequences are in grey, while other sequences from CtIP genome locus are in black. The short fragment originated from donors is highlighted with shades in light green. The number of base pairs omitted is indicated in brackets

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