10.1021/acssynbio.7b00020.s001
Chih-Che Shen
Chih-Che
Shen
Li-Yu Sung
Li-Yu
Sung
Shih-Yeh Lin
Shih-Yeh
Lin
Mei-Wei Lin
Mei-Wei
Lin
Yu-Chen Hu
Yu-Chen
Hu
Enhancing Protein Production Yield from Chinese Hamster
Ovary Cells by CRISPR Interference
American Chemical Society
2017
CRISPRi approach
G-CSF
MTX selection
CHO cell clones coexpressing DHFR
RNA
Chinese Hamster Ovary Cells
dCas 9 protein
gene
dhfr
force CHO cells
CHO cells
EGFP
CHO cell engineering
Enhancing Protein Production Yield
2017-04-18 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Enhancing_Protein_Production_Yield_from_Chinese_Hamster_Ovary_Cells_by_CRISPR_Interference/4989509
Chinese
hamster ovary (CHO) cells are an important host for biopharmaceutical
production. Generation of stable CHO cells typically requires cointegration
of <i>dhfr</i> and a foreign gene into chromosomes and subsequent
methotrexate (MTX) selection for coamplification of <i>dhfr</i> and foreign gene. CRISPR interference (CRISPRi) is an emerging system
that effectively suppresses gene transcription through the coordination
of dCas9 protein and guide RNA (gRNA). However, CRISPRi has yet to
be exploited in CHO cells. Here we constructed vectors expressing
the functional CRISPRi system and proved effective CRISPRi-mediated
suppression of <i>dhfr</i> transcription in CHO cells. We
next generated stable CHO cell clones coexpressing DHFR, the model
protein (EGFP), dCas9 and gRNA targeting <i>dhfr</i>. Combined
with MTX selection, CRISPRi-mediated repression of <i>dhfr</i> imparted extra selective pressure to force CHO cells to coamplify
more copies of <i>dhfr</i> and <i>egfp</i> genes.
Compared with the traditional method relying on MTX selection (up
to 250 nM), the CRISPRi approach increased the <i>dhfr</i> copy number ∼3-fold, <i>egfp</i> copy number ∼3.6-fold
and enhanced the EGFP expression ∼3.8-fold, without impeding
the cell growth. Furthermore, we exploited the CRISPRi approach to
enhance the productivity of granulocyte colony stimulating factor
(G-CSF) ∼2.3-fold. Our data demonstrate, for the first time,
the application of CRISPRi in CHO cells to enhance recombinant protein
production and may pave a new avenue to CHO cell engineering.