PLBD2 knockout cell line generation
In order to knock out PLBD2 gene in CHO K1 using CRISPR/Cas9, a small
guide RNA (sgRNA) sequence corresponding to Exon 1 of PLBD2 was selected
for specific targeting of PLBD2 exons 1. Sense
(5’-tgtatgagaccacGCCCCCATGGACCGGAGCCC-3’) and antisense (5’-
aaacGGGCTCCGGTCCATGGG GCgtggtctca-3’) oligonucleotides were ordered,
with appropriate overhangs for cloning into CAS940A-1 (System
Biosciences). The paired oligonucleotides were annealed at 5 µM by
incubation at 95 ºC for 5 min followed by cooling to room temperature
gradually. The annealed oligos were diluted 10x in water and ligated
into CAS940A-1 using T4 DNA ligase (ThermoFisher Scientific, Waltham,
MA). After transformation of Electromax DH10B cells (ThermoFisher
Scientific, Waltham, MA), colonies were screened by sequencing.
Maxi-preps of sequence verified plasmids containing PLBD2 sgRNA 1 was
generated using the EndoFree Plasmid Maxi Kit (Qiagen).
CHO K1 cells were transfected with 5 ug of the CAS940A-1 plasmid
encoding the PLBD2 sgRNA. The next day, the cells were harvested, and
single cell sorted into 4 96-well plates containing Optipro-SFM by flow
cytometry. After expansion, individual cell clones were analyzed by
quantitative PCR (qPCR) for the disruptions in both genomic DNA (gDNA)
and complementary DNA (cDNA). Double allelic disruption was confirmed by
PCR and sequencing. Lack of PLBD2 expression was further confirmed by
Western blot analysis.