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.