Enhancing Protein Production Yield from Chinese Hamster Ovary

Apr 18, 2017 - CRISPR interference (CRISPRi) is an emerging system that effectively suppresses gene transcription through the coordination of dCas9 pr...
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Enhancing Protein Production Yield from Chinese Hamster Ovary Cells by CRISPR Interference 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, 30013, Taiwan S Supporting Information *

ABSTRACT: Chinese hamster ovary (CHO) cells are an important host for biopharmaceutical production. Generation of stable CHO cells typically requires cointegration of dhf r and a foreign gene into chromosomes and subsequent methotrexate (MTX) selection for coamplification of dhf r 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 dhf r. Combined with MTX selection, CRISPRi-mediated repression of dhfr imparted extra selective pressure to force CHO cells to coamplify more copies of dhf r 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, egf p 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. KEYWORDS: CRISPRi, CHO cell, cell engineering, DHFR, MTX selection, protein production

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(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.7,8 CRISPR-Cas9-mediated DSB is exploited for programmable genome engineering of eukaryotic and prokaryotic 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 coexpression of dCas9 and sequence-specific 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 eukaryotic and prokaryotic cells, for rewiring metabolic

hinese hamster ovary (CHO) cells are the most common workhorse host cells for the production of approved biopharmaceutical proteins including antibodies, hormones, cytokines, and vaccines.1−4 Generation of stable CHO cell lines often hinges on the dihydrofolate reductase (DHFR)/ methotrexate (MTX) selection system for coamplification of the 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 analogue that binds and inhibits DHFR. In general, the GOI is cloned adjacent to dhf r gene in a vector and transfected into dhf r-defective CHO cells, selected with nucleoside-free medium for the cells with the GOI and dhfr cointegrated 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 a sufficient amount of DHFR can survive. The increasing MTX concentration stimulates the coamplification 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 © 2017 American Chemical Society

Received: January 22, 2017 Published: April 18, 2017 1509

DOI: 10.1021/acssynbio.7b00020 ACS Synth. Biol. 2017, 6, 1509−1519

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Figure 1. CRISPRi suppresses gene expression in CHO cells. (A) Schematic illustration of vectors. (B) dhf r expression as measured by qRT-PCR. (C) Optical and fluorescence microscopic images. (D) Flow cytometry analysis of EGFP expression. pDHFR-2A-EGFP harbored an expression cassette consisting of the CMV promoter, dhf r and egf p genes. dhf r and egf p were linked by P2A so that EGFP could be cotranslated with DHFR. pCRISPRi plasmids coexpressed dCas9-KRAB, ZeoR and gRNA. pCRISPRi-Ø expressed the scramble gRNA (ØgRNA) as a control. pCRISPRi-T expressed TgRNA to suppress the template strand (+158−177) of dhf r. pCRISPRi-NT expressed NTgRNA to target the nontemplate (+137−118) strand of dhf r. CHO DUXB11 cells were cotransfected with pDHFR-2A-EGFP and one of the pCRISPRi vectors and analyzed at 48 h posttransfection: pA, polyA signal; PU6, U6 promoter; PCMV, CMV promoter; Term, termination sequence; P2A, self-cleaving 2A peptide derived from porcine teschovirus-1.52 The data represent the averages ± SD of 3 independent culture experiments.

Figure 2. Generation of stable CHO clones coexpressing CRISPRi, DHFR, and EGFP. (A) Illustration of pCMV-EGFP-SD; (B) transfection and selection scheme for stable clone generation; (C) dCas9 expression as analyzed by qRT-PCR. The single cell clones were selected as shown (B) and described in the Results section. The dCas9 mRNA levels for each clone were measured 3 times and normalized to that in clone 2−1: PCMV, CMV promoter; PSV40, SV40 promoter.

networks21−23 and various applications (for review see ref 24). 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 coamplify dhfr and adjacent GOI. We constructed the vectors required for CRISPRi-mediated inhibition of dhfr and explored whether CRISPRi-mediated dhf r suppression can enhance dhfr amplification and transcription in stable cell clones. Whether the CRISPRi-mediated dhf r suppression concurrently increased the GOI (e.g., 1510

DOI: 10.1021/acssynbio.7b00020 ACS Synth. Biol. 2017, 6, 1509−1519

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Figure 3. EGFP expression before and after MTX selection: (A) optical and fluorescence microscopic images; (B,C) total FI before (0 nM) and after (250 nM) MTX selection. The total FI data were measured 3 times by flow cytometry for each clone, and averaged values in the same group were calculated and are shown above the bars.

enhanced green fluorescence protein (EGFP) and granulocyte colony stimulating factor (G-CSF)) expression was further evaluated.

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qRT-PCR analysis (Figure 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 dhf r, when compared with the cells transfected with pCRISPRiØ (Ø group). Fluorescence microscopy (Figure 1C) and flow cytometry (Figure 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 the CRISPRi system targeting the nontemplate strand effectively suppressed dhf r transcription (up to ∼85%) and concomitantly repressed the cotranslated EGFP (up to ∼79%). Generation of Stable CHO Clones Coexpressing CRISPRi, DHFR, and EGFP. Since pCRISPRi-NT effectively knocked down dhf r in the transient expression assay, we next assessed whether the CRISPRi-mediated dhf r 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 (Figure 2A). The dhfr-deficient CHO DUXB11 cells were transfected with pCMV-EGFP-SD and cultured for 4 weeks to select EGFPexpressing single clones. The stable clones with cointegrated dhf r and egf p genes were transfected with ZeoR-expressing pCRISPRi-Ø (Ø group) or pCRISPRi-NT (NT group) and selected using Zeocin for 2 weeks (Figure 2B). For a control mimicking the conventional method, the EGFP-expressing

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 CMV promoter, dhfr and egfp genes which were linked by a self-cleavage sequence (P2A), so that EGFP could be cotranslated with DHFR and served as a reporter (Figure 1A and Figure S1A). In parallel, we designed 3 pCRISPRi plasmids (Figure 1A and Figure S1B) harboring two expression cassettes: one coexpressing 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 nontemplate (+137−118) sequences in the dhfr gene, respectively (Figure 1A and Figure S1C). CHO cells were cotransfected with pDHFR-2A-EGFP and one of the pCRISPRi vectors and analyzed at 48 h post-transfection. 1511

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Figure 4. Transcription levels of egfp and dhf r before and after MTX selection. (A,B) Relative egf p mRNA levels at 0 nM and 250 nM MTX. (C,D) Relative dhf r mRNA levels at 0 and 250 nM MTX. All mRNA levels were measured 3 times by qRT-PCR for each clone and normalized to that of clone 2−1 at 0 nM MTX. The mRNA levels of all clones in the same group were averaged and are shown above the bars.

group were calculated. At 0 nM MTX (Figure 3B) the average total FI for the control, Ø, and NT groups was 85 ± 9, 121 ± 14, and 161 ± 23 au, respectively. After 250 nM MTX selection (Figure 3C), the average total FI in the control and Ø groups increased to 712 ± 86 and 670 ± 41 au, 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 au, which was ∼3.8-fold (p < 0.05) that in the control group, attesting that CRISPRi-mediated dhfr suppression conferred significantly more robust recombinant protein production than the traditional method. Since dhfr and egfp were designed to be coexpressed, we next analyzed the egf p and dhfr mRNA levels by qRT-PCR, using the level in clone 2−1 (at 0 nM MTX) as the baseline. Figure 4A delineates that the egf p mRNA levels were similar in all 3 groups at 0 nM MTX. After 250 nM MTX selection (Figure 4B), the egf p mRNA levels increased to 4.6 ± 0.2 and 2.6 ± 0.1 fold in the control and Ø groups, respectively, and further elevated to 8.8 ± 0.3 fold in the NT group. The dhfr mRNA levels at 0 nM MTX were similar in both the Control and Ø groups, but were significantly lower (p < 0.05) in the NT group (Figure 4C), indicating effective inhibition of dhfr transcription by CRISPRi before MTX selection. At 250 nM MTX the dhf r mRNA levels increased to 39.0 ± 1.4 and 33.5 ± 4.2 fold in the control and Ø groups, respectively, and surged to 67.1 ± 10.6 fold in the NT group (Figure 4D). After 250 nM MTX selection, the egf p and dhf r mRNA levels were statistically similar (p > 0.05) in the control and Ø groups, but were significantly (p < 0.05) enhanced in the NT group, indicating that CRISPRi-mediated targeting of dhf r

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 (Figure 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 the 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 (Figure 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 dhf r 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 Figure 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 (Figure 3A). The total fluorescence intensity (FI) of each clone was further measured by flow cytometry and average values for each 1512

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Figure 5. Gene copy numbers of egf p and dhf r before and after MTX selection. (A,B) egf p gene copy number per cell at 0 nM and 250 nM MTX. (C,D) dhf r gene copy number per cell at 0 nM and 250 nM MTX. All gene copy numbers per cell were measured 3 times by Q-PCR for each clone. The absolute gene copy numbers of all clones in the same group were averaged and are shown above the bars.

Figure 6. CRISPRi-mediated dhf r suppression did not impede CHO cell growth. (A) Growth curve. (B) Doubling time. All clones after 250 mM MTX selection were cultured and the cell numbers were measured over time. The cell numbers at the same time points for all clones in the same group were averaged. The doubling time was calculated and are shown above the bars. The data represent the averages ± SD of triplicated culture experiments.

cell, and further increased to 56.3 ± 0.5 per cell in the NT group (Figure 5B). Conversely, the dhfr gene copy numbers per cell ranged from 1.6 ± 0.2 (NT group) to 2.0 ± 0.3 (control group) at 0 nM MTX (Figure 5C), and increased to 21.3 ± 3.0, 11.3 ± 1.5, and 65.2 ± 10.2 at 250 nM MTX in the control, Ø, and NT groups, respectively (Figure 5D). At 250 nM MTX, the egf p and dhf r copy numbers were statistically similar (p > 0.05) between the control and Ø groups, indicating that the dCas9/scramble gRNA expression did not impose beneficial effects on gene amplification. Nonetheless, the NT groups conferred ∼3.6-fold and ∼3-fold (p < 0.05) higher egfp and dhfr copy numbers than

under the MTX selective pressure concomitantly ameliorated the transcription of dhf r and adjacent egf p gene, when compared with the traditional method (control group). CRISPRi-Mediated dhf r Suppression Augmented Gene Amplification. To examine whether the increased mRNA and protein levels arose from enhanced gene amplification, we analyzed the absolute copy numbers of egfp and dhfr genes per cell before and after MTX selection. Figure 5A delineates that at 0 nM MTX the average egf p copy numbers in all 3 groups were statistically similar and lower than 1. After 250 nM MTX selection, the egf p copy numbers in the control and Ø groups elevated to 12.1 ± 3.6 and 8.9 ± 1.07 per 1513

DOI: 10.1021/acssynbio.7b00020 ACS Synth. Biol. 2017, 6, 1509−1519

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Figure 7. CRISPRi-mediated dhf r suppression enhanced G-CSF expression. (A) Schematic illustration of pCMV-G-CSF-SD pA, poly A signal; PCMV, CMV promoter; Psv40, SV40 promoter. (B,C) Transcription levels of gcsf before and after MTX selection. All mRNA levels were measured 3 times by qRT-PCR for each clone and normalized to that of clone 4−1 at 0 nM MTX. The mRNA levels of all clones in the same group were averaged and are shown above the bars. (D,E) G-CSF productivity before and after MTX selection. The G-CSF productivity of all clones in the same group were averaged and are shown above the bar. The data represent the averages ± SD of triplicated culture experiments.

selected 6 clones from the G group (4−1, 4−2, 4−3, 4−4, 4−5, and 4−6) and G-NT group (5−1, 5−2, 5−3, 5−4, 5−5, and 5− 6) for subsequent selection using 50 nM and 250 nM MTX as in Figure 2B. We also confirmed the CRISPRi function during the MTX selection process as the dCas9 expression remained steady before and after MTX selection (Figure S2). The mRNA levels were measured by qRT-PCR and G-CSF secreted to the culture supernatant was quantified by ELISA and the values of different clones were averaged. Using the mRNA level of clone 4−1 at 0 nM MTX as the baseline, the average gcsf mRNA level was 1.9 ± 0.8 fold in the G group and 30.2 ± 1.1 fold in the G-NT group before MTX selection (Figure 7B). After 250 nM MTX selection, the gcsf mRNA level increased to 82.4 ± 35.0 fold in the G group and further surged to 193.0 ± 42.2 fold in the G-NT group (Figure 7C). In the protein level, the G-CSF productivity at 0 nM MTX was 0.004 ± 0.001 and 0.027 ± 0.001 pg/cell/day for the G and G-NT groups, respectively (Figure 7D). At 250 nM MTX, the G-CSF productivity rose to 071 ± 0.027 pg/cell/day in the G group and further elevated to 0.167 ± 0.023 pg/cell/day in the G-NT group (Figure 7E and Figure S3). After 250 nM MTX selection, the G-CSF expression in the G-NT group was ∼2.3fold that of the G group, in the mRNA and protein levels, confirming that CRISPRi-mediated dhfr suppression significantly enhance the G-CSF expression.

the control group, confirming that upon MTX selection CRISPRi-mediated dhf r suppression triggered the coamplification of more egf p and dhf r genes than the traditional method. CRISPRi-Mediated dhf r Suppression Did Not Affect the Cell Growth. To examine whether the CRISPRi-mediated dhfr suppression affected cell growth, we cultured all clones after 250 mM MTX selection and monitored the growth over time. The cell numbers at the same time points for all four clones in the same group were averaged. Figure 6A shows that the cells of all three groups had virtually overlapped growth curves. The doubling time in the exponential phase was 22.5 ± 0.8, 26.7 ± 1.9, and 23.8 ± 0.6 h for the control, Ø, and NT groups, without significant difference between groups (Figure 6B), indicating that the highly productive clones after CRISPRimediated dhfr suppression grew as well as the clones developed using the traditional method. CRISPRi-Mediated dhf r Suppression Enhanced G-CSF Production. After confirming the enhanced EGFP expression by CRISPRi-mediated dhf r suppression, we next exploited CRISPRi to improve the production of a pharmaceutical protein G-CSF (granulocyte colony stimulating factor). We constructed pCMV-G-CSF-SD vector which harbored the GCSF expression cassette adjacent to the DHFR cassette (Figure 7A). pCMV-G-CSF-SD was transfected into CHO DUXB11 cells, cultured for 4 weeks to select G-CSF-expressing single clones. The stable clones with cointegrated dhfr and gcsf genes were transfected with ZeoR-expressing pCRISPRi-NT (G-NT group) and selected using Zeocin for 2 weeks, or cultured in parallel without the transfection (G group). We randomly

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DISCUSSION CHO cells and the DHFR/MTX gene amplification system are routinely used to generate stable producer CHO cell clones in 1514

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ACS Synthetic Biology biopharmaceutical industries,4 but this approach is timeconsuming and laborious. In this study, we employed the CRISPRi technology to specifically knockdown dhfr transcription in attempts to enhance recombinant protein production in stable CHO cells. In conjunction with the dCas9-KRAB fusion protein, the gRNA targeting the nontemplate strand repressed dhf r more effectively than the gRNA targeting the template strand in the transient expression assay (Figure 1), which was consistent with the rule proposed previously.20,25 Furthermore, we generated stable clones with cointegrated egf p/dhfr cassettes and a functional CRISPRi system (Figure 2). Compared with the conventional MTX selection method (control group), CRISPRi-mediated suppression of dhf r coupled with MTX selection (NT group) concurrently enhanced the model recombinant protein (EGFP) expression 3.8-fold (∼2722 au) at 250 nM MTX (Figure 3). The EGFP expression could be further elevated to ∼3899 au when MTX concentration was raised to 1000 nM (Figure S4). The extent of increase from 250 nM to 1000 nM MTX was only ∼43%, probably because the excessive accumulation of intracellular EGFP provoked cellular stress and toxicity. Not only the elevated EGFP protein level, CRISPRimediated dhfr suppression also triggered higher transcription levels of dhf r and egfp after 250 nM MTX selection than the traditional method (Figure 4). This seemingly contradictory result can be ascribed to the predominant selective pressure imparted by MTX which inhibits DHFR activity. Upon the stepwise increase of MTX concentration, CRISPRi-mediated dhfr suppression imposed an additional selective pressure to attenuate the dhfr transcription, thus forcing the cells to express more DHFR to counteract the inhibitory effects of MTX in order to survive. Such extra selective pressure promoted the integration of significantly more copies of dhf r and adjacent egfp into the chromosome than the traditional method (Figure 5). The expression of dCas9 and gRNA did not disturb the cell growth (Figure 6), hence easing the concerns of decelerated cell growth and lower protein productivity. Importantly, the CRISPRi-mediated dhf r suppression was able to enhance the G-CSF expression in the mRNA and protein levels when compared with the traditional method (Figure 7). To obtain high producer CHO cells, the standard MTX amplification method may be modified by dampening dhfr expression by using a weaker promoter26 or appending destabilizing sequences such as AU-rich elements or mouse ornithine decarboxylase (MODC) PEST region on the dhfr mRNA.27 However, these methods are less flexible as the promoter or in cis elements attached to dhfr mRNA dictate the DHFR expression levels. Alternatively, dhfr mRNA levels may be knocked down by small interfering RNA (siRNA)28,29 which typically binds the 3′-UTR to block translation or induce mRNA degradation. Yet siRNA-mediated silencing requires cytoplasmic argounaute proteins and shares the same processing pathway with microRNA (miRNA). Gene silencing through siRNA may disturb the endogenous miRNA expression profile, in addition to its well-known off-target effects.30 In comparison with these methods, CRISPRi is an emerging versatile tool for programmable and customizable modulation of gene expression and requires only expression of dCas9 and the synthetic gRNA, thus avoiding the perturbation of cellular miRNA processing. Moreover, CRISPRi enables stringent suppression with one or several gRNA targeting the same gene.31 In this study we employed a single gRNA (NTgRNA)

that suppressed dhf r expression up to 85% (Figure 1). Future studies will be directed toward employing multiple gRNA targeting different regions of dhf r promoter and open reading frame to achieve tighter knockdown of dhfr transcription, which may allow us to generate producer clones with better protein yield after MTX selection. In addition, several other different approaches have been developed to enhance recombinant protein production in CHO cells, such as using stronger promoters to drive the exogenous gene,32 overexpressing cell cycle regulating protein,33 inserting matrix attachment regions34,35 and cointegrating Ubiquitin Chromatin Opening Elements36 to modify the chromatin structures. Conversely, other vectors such as transposon enables more efficient gene integration and may increase the initial copy number of dhfr and gene of interest. The CRISPRi system may be exploited in combination with these strategies to further elevate the recombinant protein yield. Furthermore, CRISPRi offers the flexibility to regulate the expression levels of multiple endogenous genes without completely abrogating the gene functions, hence representing a valuable toolkit to intricately rewire the metabolic flux in the cells.21,37 Such multiplexing capability allows one to simultaneously fine-tune the expression levels of not only dhfr, but also other genes involved in the control of cell metabolism, growth, apoptosis, and protein production, etc. For instance, disruption of C12orf35, whose gene product interferes with the nuclear export of processed mRNA,38 and knockout of IGF-1 receptor39 improve the expression of target recombinant proteins in CHO cells. CRISPRi-mediated dhfr suppression may be coupled with the regulation of these genes to redirect the metabolic networks in CHO cells so that CRISPRi can be harnessed to improve the recombinant protein production, in terms of not only yield, but also kinetics and quality. Additionally, CRISPRi can be repurposed for genome-wide screening and interrogation of gene functions24,30,40 or for targeted DNA methylation41 for epigenetic studies. With the newly unveiled CHO genome sequence,42 CRISPRi may be used in conjunction with the emerging “omics” technology2 to interrogate the functions of genes crucial for cell metabolism and product production, which will unravel more genomic, transcriptomic, and metabolomic targets for CHO cell engineering. One major concern of CRISPRi technology is the off-target effects that might perturb gene expression at off-target sites.43 In this study, the expression of dCas9 and gRNA did not appreciably mitigate the cell growth (Figure 6) yet the potential off-target effects necessitate further investigations. To minimize undesired off-target effects, the rationale of proper gRNA design has been extensively studied44,45 and several strategies to enhance the specificity (e.g., truncated sgRNA46 or Cas9 mutant with nickase activity47) have been developed. These new designs may be harnessed in future CHO cell engineering studies to minimize off-target effects. Aside from the dCas9 protein derived from S. pyogenes, other orthogonal Cas9 proteins48−50 and nuclease (e.g., Cpf151) have been discovered. These proteins differ in the specific PAM sequences and consequently can be used in the same cell when paired with their corresponding gRNA to recognize cognate targets without interfering with each other. These orthogonal proteins may be mutated to develop a catalytically inactive DNA binding protein and provide additional flexibility to engineer CHO cells by targeting genes governing transcription, translation, protein 1515

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SV40-DHFR. The egf p gene was PCR-amplified from pIRES2EGFP (Addgene) and inserted downstream of CMV promoter of pCMV-SV40-DHFR to yield pCMV-EGFP-SV40-DHFR. For simplicity, pCMV-EGFP-SV40-DHFR was abbreviated as pCMV-EGFP-SD. To integrate dhfr and gcsf into the CHO cell chromosome, we chemically synthesized human gcsf gene fused with c-myc and His tag. The synthetic gcsf gene was subcloned into the pCMV-EGFP-SD vector to replace the egfp gene to yield pCMV-G-CSF-SD. Cell Culture, Transfection, and Selection. The dhfrdeficient CHO DUXB11 cells were routinely cultured in F12 DMEM medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen). For transient dhfr suppression assay, the cells were seeded to 6-well plates (3 × 105 cells/well), cultured for 24 h, and cotransfected with pDHFR-2A-EGFP (2 μg/well) and the one of the 3 pCRISPRi plasmids (2 μg/well) using TurboFect (Thermo Fisher). The cells were analyzed at 48 h post-transfection for dhf r and EGFP expression. To generate stable clones, CHO DUXB11 cells were seeded to 6-well plates (3 × 105 cells/well), cultured using F12 DMEM medium supplemented with 10% FBS and transfected with pCMV-EGFP-SD (4 μg/well). After 24 h, the medium was changed to nucleoside-free α-MEM with 10% FBS and cells were subcultured every 3 days. After steady cell growth, single clones were generated by limiting dilution. The single cell clones were observed under the fluorescence microscope for EGFP expression. After 4 weeks of selection, 18 stable clones with cointegrated dhf r and egfp genes were picked and divided into 3 groups (Ø, NT, and control). The Ø and NT groups were seeded to 6-well plates (3 × 105 cells/well), cultured overnight using nucleosidefree α-MEM with 10% FBS and transfected with 4 μg/well of ZeoR-expressing pCRISPRi-Ø or pCRISPRi-NT, respectively. After 48 h, the transfected cells were cultured in 6-well plates using the same medium containing 100 μg/mL of Zeocin and subcultured every 3 days for 2 weeks until steady cell growth to ensure the integration of CRISPRi system. In parallel, the EGFP-expressing clones in the control group were directly cultured in nucleoside-free, Zeocin-free α-MEM without transfection. To start MTX selection, the clones in the Ø and NT groups (six clones each) were cultured in 6-well plates using nucleoside-free α-MEM containing 50 nM MTX and 100 μg/ mL Zeocin. The cells were subcultured upon 90% confluency. After 4 weeks of selection, the MTX concentration was raised to 250 nM and the selection process was repeated until cell growth stabilized. The clones in the control group were selected by a similar MTX selection scheme except that the medium was Zeocin-free. All clones before (0 nM) and after (250 nM) MTX selection were frozen in cryovials in liquid nitrogen for subsequent analyses. Fluorescence Microscopy and Flow Cytometry. The cells were observed under the fluorescence microscope (ECLIPSE TS100-F, Nikon) for green fluorescence emission. The percentage of cells emitting fluorescence and mean fluorescence intensity (FI) of each sample were measured three times by counting 10 000 cells in each measurement. Multiplying the mean FI by the number of GFP+ cells yielded the total FI. For each stable clone, the total FI was measured three times and the total FI of all clones in the same group (Ø, NT, or control) were averaged. All total FI data are expressed

folding, apoptosis, metabolic pathways, or epigenetic modifications. In summary, here we demonstrated, for the first time, effective CRISPRi-mediated repression of dhfr in CHO cells. The CRISPRi approach in conjunction with MTX selection exerted extra selective pressure to inhibit DHFR expression and activity, hence augmenting the integration and expression of DHFR and cointegrated adjacent gene of interest. This study broadens the applications of CRISPRi in CHO cells for enhanced recombinant protein production and may pave a new avenue to CHO cell engineering.

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MATERIALS AND METHODS Plasmid Construction. The pCRISPRi plasmids encoding the CRISPRi function were constructed using pUseAmp(+) (Merck Millipore) that harbored a CMV promoter as the backbone. We PCR-amplified the Zeocin resistance gene (ZeoR)-BGH poly A sequence from pSecTag2b (Thermo Fisher) and concurrently added a self-cleaving 2A peptide sequence derived from porcine teschovirus-152 with synthetic primers. The fusion PCR product (P2A-ZeoR-polyA) was subcloned into pUseAmp(+) to yield pCMV-P2A-Zeocin. We next inserted a synthetic EcoRI/XhoI restriction site downstream of the plasmid’s CMV promoter and digested dCas9KRAB (KRAB is a transcription repression domain that augments the dCas9 inhibition53) from pHAGE EF1α dCas9KRAB (Addgene) using EcoRI/XhoI. The dCas9-KRAB gene was subcloned into pCMV-P2A-Zeocin by EcoRI/XhoI treatment to yield pCMV-dCas9-KRAB-P2A-Zeocin. In parallel, the sequence encoding the U6 promoter, gRNA scaffold and terminator was PCR-amplified from pX335-U6Chimeric_BB-CBh-hSpCas9n (Addgene) and subcloned into TA vector (Invitrogen) to yield pTA-U6gRNA-Term vector. The 20 bp spacer DNA duplexes targeting the template (T) or nontemplate (NT) sequences in the dhfr gene, or targeting no sequence in dhfr (Ø) were synthesized (see Results and Figure S1), cloned into the gRNA scaffold using BbsI and sequenced to confirm the correct cloning. The resultant gRNA were designated TgRNA, NTgRNA, and ØgRNA, respectively. The U6gRNA cassette was digested using EcoRI/MfeI and subcloned into the MfeI site of pCMV-dCas9-KRAB-P2A-Zeocin. The resultant pCRISPRi plasmids (pCRISPRi-Ø encoding ØgRNA, pCRISPRi-T encoding TgRNA and pCRISPRi-NT encoding NTgRNA) contained two expression cassettes: CMV-dCas9KRAB-P2A-Zeocin and U6-gRNA. Additionally, the mouse dhf r gene was chemically synthesized (Figure S1) and cloned in between the CMV promoter and P2A sequence of pCMV-P2A-Zeocin to yield pCMVDHFR-P2A-Zeocin. The enhanced green fluorescent protein (EGFP) gene was PCR-amplified from pIRES2-EGFP (Addgene) and inserted downstream of P2A of pCMVDHFR-P2A-Zeocin, which concurrently removed the ZeoR gene. The resultant pDHFR-2A-EGFP expressed a DHFREGFP fusion protein that was autocleaved by P2A peptide and was used for transient dhf r suppression assay. To integrate both dhf r and egf p into the CHO cell chromosome, we fused the BGH polyA sequence and SV40 promoter by overlap PCR and subcloned the polyA-SV40 amplicon into pCMV-P2A-Zeocin by HindIII/XhoI treatment (and concomitantly removed P2A-Zeocin) to yield pCMVpolyA-SV40-polyA. The mouse dhfr gene was PCR-amplified from pCMV-DHFR-P2A-EGFP and inserted downstream of SV40 promoter of pCMV-polyA-SV40-polyA to yield pCMV1516

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Research Article

ACS Synthetic Biology in arbitrary units (a.u.) and represent the averages ± standard deviation (SD). Quantitative Real-Time Reverse Transcription PCR (qRT-PCR). Total RNA was isolated from CHO cells using the NucleoSpin RNA II kit (Machereye-Nagel) and reverse transcribed to cDNA using the MMLV Reverse Transcription first-strand cDNA Synthesis Kit (Epicenter Biotechnologies). The genes encoding dCas9, dhf r, and egfp were analyzed by real-time PCR (Q-PCR) using StepOnePlus Real-Time PCR Systems (Applied Biosystems) and gene-specific primers (Table S1). The β-actin expression served as an internal control. The gene expression levels were normalized to those of control cells (see Results). For each stable clone, the mRNA levels were measured 3 times and the data of all clones in the same group (Ø, NT, or control) were averaged. The relative mRNA levels represent the averages ± SD. Genomic Copy Number Quantification by Q-PCR. The genomic DNA was extracted from stable CHO cell clones using Genomic DNA mini kit (Geneaid). Q-PCR reactions were conducted with 2 μL of sample (4 ng), 0.5 μL of mixed primers (forward and reverse) specific for dhf r or egf p (Table S1), and 2.5 μL of SYBR Green PCR Master Mix (Applied Biosystems). To quantify the absolute gene copy number, the p-CMVEGFP-2A-DHFR plasmid was serially diluted (4, 0.4, 0.04, 0.004, 0.0004 μg) and quantified by Q-PCR to generate the standard curve. The absolute dhfr and egf p copy numbers per cell were then quantified based on the assumption that 6 ng total genomic DNA was equal to 1820 genomic DNA molecules.54 Analysis of G-CSF by ELISA. For each clone, the cells were seeded to 6-well plates (3 × 105 cells/mL) overnight, and medium was replaced with 2 mL of fresh α-MEM medium containing 10% FBS. After 48 h of culture, the supernatant was harvested for ELISA analysis of G-CSF using the human-GCSF DuoSet kit (R&D systems a biotechne brand DY008). The cells were trypsinized for cell number count using the hemacytometer. The G-CSF productivity was calculated as described.55 Statistical Analysis. Results are expressed as averages ± standard deviation (SD) and statistical comparisons were performed using the unpaired Student’s t-test. All data are representative of at least three independent experiments. p < 0.05 represents statistical significance.

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Author Contributions

C.C.S. designed the project, performed experiments, and wrote the paper. L.Y.S. and S.Y.L. performed experiments. Y.C.H. designed the project and wrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support from the Ministry of Science and Technology, Taiwan (MOST 105-2622-8-007-009, 104-2622-8-007-001, 103-2622-E-007-025).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00020. Vector and gRNA design; dCas9 mRNA level after 250 MTX treatment; G-CSF protein expression level after 250 nM MTX treatment; EGFP expression after 1000 nM MTX selection; primer sequences of qRT-PCR and genomic DNA Q-PCR (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: (886)3-571-8245. Fax: (886)3-571-5408. E-mail: [email protected]. ORCID

Yu-Chen Hu: 0000-0002-9997-4467 1517

DOI: 10.1021/acssynbio.7b00020 ACS Synth. Biol. 2017, 6, 1509−1519

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