Fig. 1

Inserting the sequence for the FLAG-SNAP-tag in the endogenous TERT locus. a Introducing an N-terminal FLAG-SNAP epitope tag to the endogenous TERT protein. First, a double-strand break was generated next to the translational start site of TERT with the CRISPR-Cas9 system (red scissors). Cells that underwent homologous recombination (HR) with the donor template (DT) containing the sequence for the tags and the SV40-driven enhanced green fluorescent protein (eGFP) expression cassette were screened for GFP signal and confirmed by PCR with primers a, b, c, and e, the sequences of which are listed in Table S3 in Additional file 1. HA homologous arm, TSS transcription start site. Next, the eGFP expression cassette was removed from the TERT locus through Cre-mediated recombination, leaving only the sequence for FLAG- and SNAP-tags and an intervening LoxP site at the 5′ end of the TERT coding sequence. b Fluorescence-activated cell sorting (FACS) screen for GFP-positive HEK 293 cells at the first step. Data shown are from cells transfected with the circular DT only or the circular DT + the Cas9-sgRNA plasmid. Cells with GFP signal in the green-shaded region were sorted. Inset figure: the green-shaded region enlarged. The DT-only group contained 0.5 % cells in this region and the Cas9-sgRNA + DT group contained 1.1 % cells in this region. c Sample data of PCR screen for clones that underwent HR. PCR products amplified by primer pairs a–b (upper panel) and c–e (lower panel), from genomic DNA of the GFP-positive HEK 293 single cell clones and untreated parental cells, were visualized by gel electrophoresis. Marker: 1 kb DNA ladder (Promega). Among the eight clones shown, six generated PCR products of the expected sizes in both PCRs (1402 bp for primer pair a–b, 1773 bp for primer pair c–e). Clone 2 only generated the correct PCR product in the a–b PCR. Clone 9 did not generate any PCR products, as was the case for the parental cells. d Sample data of PCR examination of the zygosity in the clones, from an experiment in which the sequence of a FLAG-tag and the eGFP expression cassette were inserted into the endogenous TERT locus in HEK 293T cells. PCR products amplified by primer pair a–e, from genomic DNA of the selected HEK 293T single cell clones and untreated parental cells, were visualized by gel electrophoresis. PCR with genomic DNA from the parental cells only generated the expected 2384 bp PCR product. Clones carrying the targeted insertion in both TERT alleles should generate only the 3652 bp PCR product. Clones carrying the targeted insertion in one of the TERT alleles should generate PCR products of both lengths. All eight clones shown were heterozygous for the insertion. The middle bands (indicated by asterisk) between the 3652 and 2384 bp bands might be the hybrid of one 2384 nucleotide strand and one 3652 nucleotide strand, since it contains the same sequence as the top band corresponding to the edited allele in our sequencing data. The same experiment was later performed on the HEK 293 clones with the insertion of FLAG-SNAP-tag sequence and the eGFP-expression cassette. The PCR product from the allele that underwent HR should be 4321 bp, which we failed to obtain, presumably because of the long length. We then regarded a clone as heterozygous if the 2384 bp product appeared. e FACS screen for GFP-negative cells at the second step. After transfection of the Cre plasmid or no transfection, cells with low GFP signal (blue-shaded region) were sorted. f Sample data of PCR screen for clones in which the eGFP expression cassette was excised through Cre recombination. PCR products amplified by primer pair a–d, from genomic DNA of the original GFP-positive clone without Cre expression (Cre ⁻) and several single cell GFP-negative clones after Cre recombination (Cre ⁺), were visualized by gel electrophoresis. Marker: 1 kb DNA ladder (Promega). In Cre⁻ cells, the size of the PCR product is 3031 bp. After the eGFP expression cassette is excised by Cre recombination, the size of the PCR product decreases to 1874 bp