Figure legends

Figure 1. The occurrence of CRISPR/Cas9-mediated targeted integration was analyzed using promoter-trap based CHO-K1 monitoring cell line. (a) Schematic outline of the promoter-trap based CHO-K1 monitoring cell line. The monitoring cell line contains the promoter-less EGFP expression cassette in the non-coding region. After co-transfection of Cas9/sgRNA expression vector and promoter donor targeting the EGFP upstream region, EGFP expressing cells can be generated by the HDR pathway. (b) Analysis of knock-in efficiency of three different promoters. The promoter-trap based monitoring CHO-K1 cell lines were co-transfected with Cas9 or Cas9/ sgRNA1 expression vector and three exogenous constitutive promoter donors including a short variant of CMV, EF-1α and CHEF-1α promoter. The EGFP+ percentage (%) was determined by flow cytometry three and six days after electroporation. (c) Mean fluorescence intensity (MFI) of the EGFP+ populations on day 6. MFI reflects the different strength of knock-in promoters. In (b) and (c), results are shown as the average values ± standard error of the mean (SEM) in three independent experiments. (d) 5’ Junction PCR validation of EGFP+ sorting pool cells. The transfected pool of cells were FACS sorted for EGFP+ populations, followed by genomic DNA purification. Promoter specific primer sets were used to amplify each 5’ Junction region. The expected amplicon size of short variant of CMV (lane 2), EF1α promoter (lane 4) and CHEF1α promoter (lane 5) was 1313 bp, 1389 bp and 1841 bp respectively. The mock was no DNA control (lane 1,3).
Figure 2. Comparison of conventional circular donor (CCD) and double cut donor (DCD) on knock-in efficiency. (a) Schematic description of conventional CCD and DCD. The CCD is normally used plasmid donor, whereas DCD is a modified form where both 5’ and 3’ homology arms are flanked by two sgRNA recognition sequences which are identical to the target genomic sgRNA/PAM sequence. Comparison of knock-in efficiency of CCD and DCD in (b) adherent monitoring cell lines and (c) in serum-free suspension adapted monitoring cell lines. Cas9 or Cas9/sgRNA1 expression vector and three different promoter donors (CCD or DCD) were co-transfected to the promoter-trap based monitoring CHO-K1 cell line. The EGFP+ percentage (%) was measured by flow cytometry three and six days after electroporation. In (b) and (c), results are shown as the average values ± SEM in three independent experiments, and the representative histograms are shown.
Figure 3. Generation of double-targeted integrants using double cut donors (DCDs) in the simultaneous and sequential knock-in (KI) strategies. (a) Schematic outline of double-KI monitoring CHO-K1 cell line. The promoter-less EGFP and TagRFP657 cassettes were integrated into the non-coding region (site 1) and COSMC locus (site 2), respectively. The second monitoring cassette includes mouse Rosa26 sgRNA targeting sequence upstream of TagRFP657 coding sequence. Co-transfection with the Cas9/sgRNA1 expression vector and donor plasmid targeting site 1 and/or Cas9/sgRNA_mouse Rosa26 expression vector and donor plasmid targeting site 2, results in single or double targeted integration. (b) Two strategies for double targeted integration. The simultaneous double-KI strategy delivers all vector constructs into cells through a single transfection process. (Cas9/sgRNA1, Cas9/sgRNA_mouse Rosa26, EF1α donor targeting non-coding region, and EF1α donor targeting COSMC locus). The sequential double-KI strategy creates double-KI pool cells by one by one targeting at 6-day time intervals through two rounds of targeted integration (Cas9/sgRNA1 and EF1α donor targeting non-coding region for site 1 and Cas9/sgRNA_mouse Rosa26 and EF1α donor targeting COSMC locus for site 2 independently). Flow cytometry analysis of double-KI efficiency in (c) the sequential KI strategy and (d) the simultaneous KI strategy. The EGFP+ percentage (%) was determined by flow cytometry on day 6 and/or day 12. In (d), both adherent and suspension monitoring cell lines were used. In (c) and (d), results are shown as the average values ± SEM in three independent experiments, and the representative dot plots are shown.