# The score describes amounts of databases that predict the
corresponding gene target of a miRNA out of 12 selected database in
miRWalk.
These targets were examined further below.
KO/KD cell lines
In order to test the effect of reduced/loss-of-function miRNAs for three
specific miRNAs – miR-128, miR-34C, and miR-181, which all have
potential targets in glycosylation – and evaluate possible correlation
between selected miRNAs and N-glycosylation, we set up a CRISPR-based
engineering strategy.
For each target, two plasmids were generated, each expressing one
specific guide RNA (gRNA) targeting the upstream and downstream region
of the miRNA expression site in the CHO genome (up-sgRNA and dw-sgRNA
respectively).
For each target, up-sgRNA and dw-sgRNA plasmids were co-transfected into
CHO-S cells, and subsequently single cell sorting and screened. As shown
in Figure 2A , KO/KD of miRNA was first verified at the molecular level
by genomic DNA PCR. It was possible to isolate both miR-128, miR-34C,
and miR-181 KO/KD clones which showed a clean deletion (KO). For some
miR-128 and miR-34c clonal cell lines, both wt bands and KO bands were
found in the gel. This indicates that the deletion may only have
happened in one allele in these cell lines, thus making them KD rather
than KO, and suggesting that these exist in more than one allele.