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Enhancing Protein Production Yield from Chinese Hamster Ovary Cells by CRISPR Interference
journal contribution
posted on 2017-04-18, 00:00 authored by Chih-Che Shen, Li-Yu Sung, Shih-Yeh Lin, Mei-Wei Lin, Yu-Chen HuChinese
hamster ovary (CHO) cells are an important host for biopharmaceutical
production. Generation of stable CHO cells typically requires cointegration
of dhfr and a foreign gene into chromosomes and subsequent
methotrexate (MTX) selection for coamplification of dhfr 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 dhfr transcription in CHO cells. We
next generated stable CHO cell clones coexpressing DHFR, the model
protein (EGFP), dCas9 and gRNA targeting dhfr. Combined
with MTX selection, CRISPRi-mediated repression of dhfr imparted extra selective pressure to force CHO cells to coamplify
more copies of dhfr and egfp genes.
Compared with the traditional method relying on MTX selection (up
to 250 nM), the CRISPRi approach increased the dhfr copy number ∼3-fold, egfp 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.