Fig 2: 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 CtIP5′UTR −/− clones. Cells transiently transfected with CtIP cDNA were included as positive control. OE, overexpression. e Sequences of the RT-PCR products amplified from aberrant CtIP5′UTR transcripts with primers CtIP_F5/R5* in c. Shown are junction sequences of two aberrant CtIP5′UTR 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

Fig 3: Targeted knock-in of pgk-GFP/Td reporters at CtIP exon-7 yielded cells carrying distinct in-frame transcripts. a Schematic for dual reporter knock-in at CtIP using pgk-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. Numbers of single-cell clones analyzed and success rates of complete disruption are shown. b Genome PCR showing CtIP disruption, forward and reserve integrations of donors, in selected CtIPE7pgk -/- clones raised in a. c RT-PCR analysis of the selected CtIPE7pgk -/- clones. Shown are schematic of CtIP mRNA, sgCtIP target site, and primer binding positions (top), gel electrophoresis of RT-PCR products (middle), and quantitative RT-PCR analysis using primers CtIP_F3/R3 (bottom) (Additional file 2: Individual data values for Fig. 4c). d Western blot analysis of the selected CtIPE7pgk -/- clones using antibodies against CtIP and ß-actin. Cells transiently transfected with CtIP cDNA were included as positive control. OE, overexpression. e Junction sequences of two aberrant CtIPpgk transcripts amplified using primers CtIP_F2/R2 in c. The sg-A target sequence is 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. Three short fragments originated from donors are highlighted with shades in different colors. The numbers of base pairs omitted are indicated in brackets. f Schematic for the modified CtIP allele harboring reversely integrated pgk-GFPdonor or pgk-Tddonor (top). Red bars below indicated the positions of sequences detected in the aberrant CtIPpgk transcripts. Sequences showing cryptic splice sites and the splicing events involved in producing the aberrant CtIPpgk transcripts detected (bottom)