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Enhancing Protein Production Yield from CHO Cells by CRISPR Interference (CRISPRi) Chih-Che Shen, Li-Yu Sung, Shih-Yeh Lin, Mei-Wei Lin, and Yu-Chen Hu ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 18 Apr 2017 Downloaded from http://pubs.acs.org on April 19, 2017
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Enhancing Protein Production Yield from CHO Cells by CRISPR Interference (CRISPRi)
Chih-Che Shen, Li-Yu Sung, Shih-Yeh Lin, Mei-Wei Lin and Yu-Chen Hu* Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan 30013
Running Title: CRISPRi to enhance protein yield in CHO cells
*Corresponding Author Phone: (886)3-571-8245 FAX: (886)3-571-5408 Email:
[email protected] 1
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Abstract CHO cell is an important host for biopharmaceutical production. Generation of stable CHO cell typically requires co-integration of dhfr and foreign gene into chromosomes and subsequent methotrexate (MTX) selection for co-amplification 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 co-expressing 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 co-amplify 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 for ≈3-fold, egfp copy number for ≈3.6fold and enhanced the EGFP expression for ≈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) for ≈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.
Keywords: CRISPRi, CHO cell, cell engineering, DHFR, MTX selection, protein production
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Key points
We proved, for the first time, effective CRISPRi-mediated gene suppression in
CHO cells.
CRISPRi-mediated dhfr repression augmented co-amplification of dhfr and
foreign gene during MTX selection.
CRISPRi-mediated dhfr repression improved recombinant protein production in
CHO cells.
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INTRODUCTION Chinese hamster ovary (CHO) cells are the most common workhorse host cells for the production of approved biopharmaceutical proteins including antibodies, hormones, cytokines and vaccines.14
Generation of stable CHO cell lines often hinges on the dihydrofolate reductase
(DHFR)/methotrexate (MTX) selection system for co-amplification of dhfr gene and the gene of interest (GOI). DHFR is an enzyme converting folate to tetrahydrofolate and is crucial for biosynthesis of glycine, purines and thymidylic acid. MTX is a folate analog that binds and inhibits DHFR. In general, the GOI is cloned adjacent to dhfr gene in a vector and transfected into dhfr-defective CHO cells, selected with nucleoside-free medium for the cells with the GOI and dhfr co-integrated into the chromosome, followed by further selection with stepwise increase of MTX concentration. Since MTX inhibits DHFR, the cells compensate the inhibition through dhfr amplification and only cells expressing sufficient amount of DHFR can survive. The increasing MTX concentration stimulates the co-amplification of dhfr and GOI, hence increasing the copy number of GOI in the chromosome and enhancing the GOI expression level.5 However, this process is a major bottleneck in CHO cell engineering as numerous rounds of selection and hence several months are required to obtain cells with high gene copy numbers.5 CRISPR-Cas9 is a newly developed RNA-guided genome editing system6 comprising the Cas9 nuclease, transacting RNA (tracrRNA) and CRISPR RNA (crRNA). Guided by the spacer sequence on crRNA, the Cas9/crRNA/tracrRNA complex orchestrates to recognize the protospacer-adjacent motif (PAM) on the chromosome and bind to proximal complementary sequence, thereby triggering a double strand break (DSB) at the target sequence.6 The system is simplified by using a chimeric guide RNA (gRNA) composed of the mature crRNA fused to tracrRNA to mimic the natural crRNA:tracrRNA duplex.7,
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CRISPR-Cas9-mediated DSB is
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exploited for programmable genome engineering of eucaryotic and procaryotic cells,9-11 as well as for gene and cell therapy.12-14 Furthermore, CRISPR-Cas9 has been used to engineer the genome of CHO cells by knocking in/out genes related to the product yield and/or quality.15-19 In addition, the catalytic domains of Cas9 are mutated to generate the catalytically inactive Cas9 (dCas9) for CRISPR interference (CRISPRi). After co-expression of dCas9 and sequencespecific gRNA, the dCas9/gRNA complex binds to the promoter or open reading frame of target gene and blocks the RNA polymerase binding/movement, hence repressing target gene transcription initiation/elongation.20 CRISPRi was recently repurposed to repress genes in a wide variety of eucaryotic and procaryotic cells, for rewiring metabolic networks21-23 and various applications (for review see reference24). However, whether CRISPRi functions in CHO cells has yet to be demonstrated. In this study, we hypothesized that CRISPRi can be harnessed to suppress dhfr gene transcription upon the MTX selection process so as to impart extra selective pressure and force the cells to co-amplify dhfr and adjacent GOI. We constructed the vectors required for CRISPRimediated inhibition of dhfr and explored whether CRISPRi-mediated dhfr suppression can enhance dhfr amplification and transcription in stable cell clones. Whether the CRISPRimediated dhfr suppression concurrently increased the GOI (e.g. enhanced green fluorescence protein (EGFP) and granulocyte colony stimulating factor (G-CSF)) expression was further evaluated.
RESULTS Evaluation of CRISPRi-mediated gene knockdown in CHO DUXB11 To evaluate the effectiveness of CRISPRi for repressing gene expression in CHO DUXB11 cell line, we first constructed pDHFR-2A-EGFP that harbored an expression cassette consisting of 5
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CMV promoter, dhfr and egfp genes which were linked by a self-cleavage sequence (P2A), so that EGFP could be co-translated with DHFR and served as a reporter (Fig. 1A and Fig. S1A). In parallel, we designed 3 pCRISPRi plasmids (Fig. 1A and Fig. S1B) harboring two expression cassettes: one co-expressing dCas9 fused with the transcription repression domain KRAB and Zeocin resistance gene (ZeoR), while the other expressing different gRNA. The scramble gRNA (ØgRNA) in pCRISPRi-Ø targeted no sequences in pDHFR-2A-EGFP as a control; while TgRNA in pCRISPRi-T and NTgRNA in pCRISPRi-NT targeted the template (+158∼177) and non-template (+137∼118) sequences in the dhfr gene, respectively (Fig. 1A and Fig. S1C). CHO cells were cotransfected with pDHFR-2A-EGFP and one of the pCRISPRi vectors and analyzed at 48 h posttransfection. qRT-PCR analysis (Fig. 1B) showed that the cells transfected with pCRISPRi-T (T group) and pCRISPRi-NT (NT group) expressed only 34.6%±4.3% and 15.2%±0.4% of dhfr, when compared with the cells transfected with pCRISPRi-Ø (Ø group). Fluorescence microscopy (Fig. 1C) and flow cytometry (Fig. 1D) illustrated that the T and NT groups expressed only 50.0±1.6% and 21.6%±2.8% of EGFP relative to the Ø group. These data confirmed that CRISPRi system targeting the non-template strand effectively suppressed dhfr transcription (up to ≈85%) and concomitantly repressed the co-translated EGFP (up to ≈79%). Generation of stable CHO clones co-expressing CRISPRi, DHFR and EGFP Since pCRISPRi-NT effectively knocked down dhfr in the transient expression assay, we next assessed whether the CRISPRi-mediated dhfr suppression could enhance recombinant protein production in stable clones. To this end, we constructed pCMV-EGFP-SD which accommodated the EGFP (as a model protein) expression cassette adjacent to the DHFR expression cassette (Fig. 2A). The dhfr-deficient CHO DUXB11 cells were transfected with pCMV-EGFP-SD and 6
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cultured for 4 weeks to select EGFP-expressing single clones. The stable clones with cointegrated dhfr and egfp genes were transfected with ZeoR–expressing pCRISPRi-Ø (Ø group) or pCRISPRi-NT (NT group) and selected using Zeocin for 2 weeks (Fig. 2B). For a control mimicking the conventional method, the EGFP-expressing stable clones were cultured in parallel without transfection (Control group). To assess the dCas9 integration and expression, we picked 4 single clones with stable EGFP expression from each group and analyzed dCas9 transcription by qRT-PCR (Fig. 2C). Compared with one clone in the Ø group (clone 2-1), the 4 clones in the Control group (clones 1-1, 1-2, 1-4, 1-5) expressed no dCas9 whereas clones in the Ø (clones 2-1, 2-2, 2-4, 2-5) and NT (clones 3-1, 3-3, 3-4, 3-6) groups expressed dCas9, albeit at fluctuating levels, demonstrating that CRISPRi system was successfully integrated into stable clones in the Ø and NT groups. Therefore, we continued to culture these clones in nucleoside-free α-MEM medium with 50 nM MTX for 4 weeks and 250 nM MTX for another 4 weeks for gene amplification (Fig. 2B). During the MTX selection, the medium was supplemented with Zeocin to ensure that CRISPRi continued to function. The clones in the Control group were selected with MTX in the same manner, but without Zeocin. CRISPRi-mediated dhfr suppression enhanced recombinant protein expression To evaluate the effects of CRISPRi-mediated targeting of dhfr during MTX selection process, the clones before (0 nM) and after (250 nM) MTX selection were observed under the microscope. As illustrated in Fig. 3A, no remarkable differences in EGFP expression existed between clones and between groups at 0 nM MTX, yet all 3 groups expressed apparently more EGFP after 250 nM MTX selection, Notably, the EGFP expression appeared similar in the Control and Ø groups but was much stronger in the NT group (Fig. 3A). 7
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The total fluorescence intensity (FI) of each clone was further measured by flow cytometry and average values for each group were calculated. At 0 nM MTX (Fig. 3B) the average total FI for the Control, Ø and NT groups were 85±9, 121±14 and 161±23 a.u., respectively. After 250 nM MTX selection (Fig. 3C), the average total FI in the Control and Ø groups increased to 712±86 and 670±41 a.u., without significant difference (p>0.05) between these two groups, indicating that expression of dCas9 and scramble gRNA (Ø) did not enhance or mitigate the recombinant protein production upon MTX selection. In contrast, the average total FI in the NT group rose to 2722±632 a.u., which was ≈3.8-fold (p
