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.