CRISPR-Cas13d for CHO Cell Engineering and Antibody Production
Mei-Wei Lin1,2, Chih-Che Shen1,
Yen-Ju Lin2, Min-Yuan Chou2, Nam
Ngoc Pham1, Yi-Hao Chang1, Chin-Wei
Chang1, Jih Ru Hwu3,4 and Yu-Chen
Hu1,4*
1Department of Chemical Engineering, National Tsing
Hua University, Hsinchu, Taiwan.
2Biomedical Technology and Device Research
Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan.
3Department of Chemistry, National Tsing Hua
University, Hsinchu, Taiwan.
4 Frontier Research Center on Fundamental and Applied
Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan.
Running Title: CRISPR-Cas13d for CHO engineering
*Corresponding Author
Phone: (886)3-571-8245
FAX: (886)3-571-5408
Email: yuchen@che.nthu.edu.tw
Abstract
Chinese hamster ovary (CHO) cell is the predominant cell factory to
produce biopharmaceuticals such as immunoglobulin G (IgG), but in CHO
cells lactate accumulation and cell death compromise IgG production
while fucosylation mitigates the antibody-dependent cellular
cytotoxicity (ADCC) of IgG. To tackle these problems, we harnessed a
burgeoning gene silencing system, CRISPR-Cas13d, to effectively suppress
the endogenous genes governing lactate production (LDHA), fucosylation
(GFT) and cell death (DDIT3), hence repressing lactate accumulation and
core fucosylation. We further exploited the Sleeping Beautysystem to integrate the CRISPR-Cas13d module co-targeting these 3 genes
and generated a CHO cell platform for IgG production. The new platform
exhibited simultaneous knockdown of LDHA, GFT and DDIT3, accumulated
less lactate, had prolonged longevity, produced more IgG with less
fucosylation and stronger ADCC efficacy. These data collectively warrant
the potentials of CRISPR-Cas13d for CHO cell engineering and improving
antibody production with regard to quantity and quality.
Keywords: antibody production, Cas13d, CRISPR, CHO, gene
knockdown
Introduction
Monoclonal antibody (mAb) is the predominant type of biopharmaceutical
anti-cancer drug (1 ). Most mAb in clinical use are immunoglobulin
G (IgG) whose glycosylation pattern dictates the efficacy and safety of
IgG as an anti-cancer drug. Such requirements for glycosylation render
Chinese hamster ovary (CHO) cell the predominant cell chassis for IgG
production (2 ). During the CHO cell culture and IgG production,
however, lactate is produced and accumulates, thus negatively impacting
on protein production (3, 4 ). Meanwhile, massive production of
misfolded or unfolded IgG provokes endoplasmic reticulum (ER) stress and
hence triggers CHO cell death (2 ). Conversely, antibody-dependent
cellular cytotoxicity (ADCC) is a key mechanism for antibody-mediated
killing of cancer cells (5 ) and requires interactions between the
fragment crystallizable (Fc) region of IgG and FcγIIIa receptors on
immune cells (e.g. natural killer cells). Fucosylation of IgG at the Fc
region hinders the interactions and compromises ADCC activity
(6 ). However, recombinant IgG produced from CHO cells are heavily
fucosylated (7 ). Removal of fucose from these oligosaccharides
enhances ADCC of antibodies such as Herceptin(8 ).
LDHA encodes the subunit A of lactate dehydrogenase which converts
pyruvate to lactate, resulting in lactate accumulation along CHO cell
culture and reducing recombinant protein yield (9 ). Fucosylation
requires the transport of GDP-fucose into Golgi apparatus by GDP-fucose
transporter (GFT) (6 ). Damage-inducible transcript 3 (DDIT3)
encodes C/EBP homologous protein and is induced by ER stress, hence
triggering cell apoptosis (10 ). As such, inhibiting endogenous
expression of LDHA, GFT and DDIT3 may offer a new strategy to improve
antibody production in CHO cells.
CRISPR is the innate immune system in prokaryotic cells to defend
against invading nucleic acids, and has been repurposed for DNA and RNA
editing (11, 12 ). Type II CRISPR system comprising Cas9 and guide
RNA (gRNA) has been leveraged to target sequence-specific DNA for genome
engineering in CHO cells (13, 14 ). Recently, type VI CRISPR
system encompassing such effectors as Cas13a, Cas13b and Cas13d
(15-18 ) was also discovered. These effectors coordinate with the
single CRISPR RNA (crRNA) to target specific single stranded RNA (ssRNA)
for cleavage (19 ) and have been harnessed for gene knockdown
(15-18 ). Among these effectors, Cas13d from Ruminococcus
flavefaciens strain XPD3002 is the most effective for gene silencing
(18 ) and can process crRNA and ssRNA. Cas13d binds to precursor
crRNA transcripts and cleaves them to produce mature crRNA composed of a
5’ direct repeat and a 3’ spacer that targets the ssRNA. Cas13d
associates with crRNA and specifically binds the ssRNA with sequence
complementary to the spacer, leading to Cas13d-mediated ssRNA cleavage
(20 ). Ectopic co-expression of Cas13d and the engineered mature
crRNA (denoted as gRNA) knocks down target mRNA with high efficiency and
high specificity without significant off-target effects in HEK293 cells
(18 ). Very recently, CRISPR-Cas13d-mediated gene downregulation
was exploited for neural cell fate manipulation (21 ), liver
metabolism regulation (22 ) and neovascularization prevention
(23 ) in mice.
Due to the roles of LDHA, GFT and DDIT3, we hypothesized that
simultaneous inhibition of these 3 genes can prolong the cell longevity
along CHO cell culture and improve the antibody production. We chose to
explore CRISPR-Cas13d to knockdown these 3 genes, individually or in
combination. Although CRISPR-Cas13d has been exploited in other
mammalian cells, whether CRISPR-Cas13d can efficiently and stably
suppress endogenous gene expression and alter the CHO cell performance
remains to be explored. In this study we demonstrated that CRISPR-Cas13d
was able to efficiently knockdown these 3 genes. We further showed that
CRISPR-Cas13d-mediated silencing altered the CHO phenotype, as well as
augmented the antibody production with regard to quantity and quality.
Results
To explore the feasibility of using CRISPR-Cas13d to silence LDHA, GFT
and DDIT3 in CHO cells, we first tested the gRNA design and constructed
a series of pCas13d-gRNA plasmids that co-expressed Cas13d and one gRNA
for each gene (Fig. 1A). The gRNA was composed of the 5’ direct repeat
and 3’ spacer that targeted different sites on the coding sequences
(Fig. 1B). We predicted 3 sites with the highest targeting scores on the
transcripts of each gene (Fig. S1) and designed 3 gRNA for LDHA and
DDIT3 (Figs. 1B-1C). Yet the 3 predicted target sites for GFT were in
close proximity (Fig. S1), thus we designed only one gRNA for GFT. As a
control, we also constructed pCas13d-gRNAØ that
expressed Cas13d and the scramble gRNAØ consisting of
the direct repeat only (Fig. 1B).
We transfected the plasmids separately into CHO cells and analyzed the
gene expression at 2 days post-transfection. Compared with
gRNAØ, the 3 gRNA for LDHA (gRNALDHA-g1to gRNALDHA-g3) significantly repressed LDHA expression,
with gRNALDHA-g1 most efficiently knocking down the mRNA
level to ≈12% (Fig. 1D). The gRNA targeting GFT also enabled effective
knockdown of GFT to 27% (Fig. 1E). For DDIT3, only
gRNADDIT3-g3 suppressed the expression to 72% while
gRNADDIT3-g1 and gRNADDIT3-g2 failed to
knock down DDIT3 (Fig. 1F), thus underscoring the importance of gRNA
design. These data demonstrated the feasibility of CRISPR-Cas13d for
gene knockdown in CHO cells. The gRNAs conferring the most effective
knockdown were selected for subsequent experiments.
Chromosomal integration of CRISPR-Cas13d module conferred stable gene
knockdown and altered CHO cell phenotype
We next aimed to integrate the entire CRISPR-Cas13d system into the
chromosome to generate stable cells. CRISPR-Cas13d system comprises
Cas13d, gRNA and the selectable marker. However, pCas13d-gRNA plasmids
(Fig. 1) did not guarantee the co-integration of Cas13d and gRNA with
the selectable marker, which could result in a heterogeneous population
of cells with incomplete CRISPR-Cas13d module. To ensure the
co-integration of Cas13d and gRNA, we exploited the Sleeping
Beauty (SB) system, which uses the transposase SB100X to transpose the
gene cassette flanked by IR/DR (inverted repeat/direct repeat) sequences
into the chromosome (24 ). We constructed pIS-gRNA plasmids (Fig.
2A) that expressed the codon-optimized SB100X and CRISPR-Cas13d module
(gRNA, Cas13d and Zeocin resistance gene (ZeoR) that
were linked by a P2A sequence). The CRISPR-Cas13d module was flanked by
a pair of IR/DR sequences to ensure co-integration of the system. The
plasmids expressing the scramble gRNAØ (pIS-Ø) or gRNA
targeting LDHA (pIS-LDHA), GFT (pIS-GFT) or DDIT3 (pIS-DDIT3) were
separately transfected into CHO cells, followed by selection using
Zeocin for 7 passages to generate stable cells.
Compared with the control pIS-Ø, pIS-LDHA significantly knocked down
LDHA expression to 55% (Fig. 2B) and significantly (p<0.05)
reduced lactate production (Fig. 2C). Meanwhile, pIS-GFT significantly
repressed GFT expression to 47% (Fig. 2D). To attest whether GFT
knockdown reduced fucosylation, we performed Lens culinarisagglutinin (LCA) assay because LCA lectin preferentially binds to the
core fucose of N-glycans and causes cell death if the cell is expressing
core-fucosylated proteins (25, 26 ). As shown in Fig. 2E, pIS-GFT
gave rise to significantly (p<0.05) higher viability than
pIS-Ø at high LCA concentrations, indicating that GFT knockdown
repressed core fucosylation and enhanced the cell tolerance to LCA.
Moreover, pIS-DDIT3 significantly repressed the DDIT3 mRNA levels to
22% (Fig. 2F) and reduced the DDIT3 protein levels at 2 and 6 days
after subculture, as demonstrated by intracellular immunostaining (Fig.
2G). These data altogether confirmed that the SB system, in conjunction
with CRISPR-Cas13d, enabled the generation of stable pools which knocked
down selected gene and conferred desired cell phenotype. Intriguingly,
the stable selection resulted in lower degrees of LDHA and GFT
inhibition than transient transfection (Fig. 1), probably because the
copy number of CRISPR-Cas13d modules was higher at 2 days after
transfection than after Zeocin selection and integration.
CRISPR-Cas13d crRNA array enabled multiplexing knockdown
To assess the efficacy of simultaneous knockdown of LDHA, GFT and DDIT3,
we constructed pIS-LGD that resembled pIS-gRNA but expressed a crRNA
array that co-targeted LDHA, GFT and DDIT3 (Fig. 3A). pIS-LGD and pIS-Ø
were individually transfected into CHO cells, and selected with Zeocin
for 7 passages to yield the stable cells IS-LGD and IS-Ø, respectively.
qRT-PCR analysis revealed that, in comparison with IS-Ø, IS-LGD
expressed similar levels of Cas13d (Fig. 3B), but significantly and
concurrently repressed LDHA, GFT and DDIT3 (Figs. 3C-3E).
To examine whether this engineered cell line can serve as a platform for
enhanced IgG production, we used a plasmid expressing a biosimilar IgG
that resembled Herceptin, an IgG drug for the
treatment of HER2-rich breast cancer (26 ). The plasmid was
transfected into the parental CHO cells, IS-Ø or IS-LGD, followed by
selection for 10 passages to yield different stable cells expressing the
Herceptin-like IgG (designated as Parental-H, IS-Ø-H
and IS-LGD-H, respectively). After subculture, the cells were cultured
for 3 days and the IgG titers were measured for the analysis of specific
production rate (SPR). As shown in Fig. 3F, the SPR of IS-Ø-H was
slightly higher than that of Parental-H but the difference was
insignificant (p>0.05), suggesting that expression of
Cas13d and gRNAØ did not deter the IgG production.
Instead, the SPR of IS-LGD-H was significantly (p<0.05) higher
than those of Parental-H and IS-Ø-H, attesting that
CRISR-Cas13d-mediated knockdown of LDHA, GFT and DDIT3 enhanced the IgG
production.
CRISPR-Cas13d-mediated knockdown improved the cell phenotype and IgG
production
To assess how the CRISPR-Cas13d-mediated gene knockdown affected the
cell response to survival stress, we cultured the engineered (IS-LGD-H
or IS-Ø-H) and parental (Parental-H) CHO cells in a batch mode until the
viability dropped below or near 50%. As shown in Fig. 4, the Parental-H
cells underwent exponential growth from day 0 to day 5 as the viable
cell density (VCD) increased exponentially (Fig. 4A) and the viability
remained higher than 90% (Fig. 4B). In parallel, the glucose
concentration decreased sharply (Fig. 4C) and lactate rapidly
accumulated (Fig. 4D). From day 5 to day 7, the Parental-H cells entered
the stationary phase as judged from the stagnant VCD and decreasing
viability, which was concurrent with the depletion of glucose and
decreasing lactate concentration, indicating that the cells were using
lactate as an alternative carbon/energy source in lieu of glucose
(9 ). At day 8, the viability precipitously dropped to ≈5% and
both glucose and lactate concentrations were low, thus the culture was
terminated.
For the IS-Ø-H cells, the exponential growth phase lasted until day 4
after which time the VCD (Fig. 4A) and viability (Fig. 4B) dropped
sharply. Due to the cell death, the culture was terminated at day 7. In
comparison with the Parental-H cells, the glucose level decreased
similarly fast (Fig. 4C) and was accompanied by lactate accumulation.
The lactate level only slightly decreased from day 5 to day 7, due to
cell death and retarded cell metabolism. These data indicated that the
culture condition became more adverse along culture time due to nutrient
depletion and lactate accumulation, hence resulting in cell death.
In contrast, the IS-LGD-H cells grew exponentially in the first 5 days
and then entered the stationary phase from day 5 to day 8 at which time
the viability remained higher than 85% (Figs. 4A-4B). Compared with the
Parental-H and IS-Ø-H cells, the IS-LGD-H cells grew more slowly (Fig.
4A), but had a longer stationary phase and the viability remained higher
than 50% even at day 10 (Fig. 4B), indicating that the
CRISPR-Cas13d-mediated silencing increased the longevity of CHO cells.
In parallel, the glucose consumption was slower (Fig. 4C) and the
lactate concentration of the IS-LGD-H cells was significantly lower than
the control IS-Ø-H cells at days 2 and 3 (Fig. 4D). The lactate
concentration remained steady from day 4 to day 10 probably because the
glucose was not depleted until day 9, thus the cells continued to use
glucose as the major carbon source and did not consume lactate during
the stationary phase (9 ).
Despite the slower growth, IS-LGD-H cells gave rise to ≈47% and 59%
higher IgG titer than IS-Ø-H and Parental-H cells, respectively (Fig.
4E), demonstrating that simultaneous silencing LDHA, GFT and DDIT3
improved the IgG production.
CRISPR-Cas13d-mediated knockdown improved the IgG quality
To ensure the IgG was correctly glycosylated and functional, we first
performed LCA sensitivity assay (Fig. 5A) as LCA kills cells with core
fucosylation (27 ). At high LCA concentrations (25 and 50 µg/ml),
the viability of IS-LGD-H was significantly higher than those of IS-Ø-H
and Parental-H, indicating that IS-LGD-H conferred less core
fucosylation of IgG and hence was more resistant to LCA.
Moreover, the IgG was purified and subjected to ADCC reporter assay to
validate ADCC efficacy of IgG produced from the engineered cells. In the
ADCC reporter assay, more efficient IgG binding to the target cells
recruits the recombinant effector cells and triggers higher luciferase
expression. As shown in 5B, the IgG from IS-LGD-H gave rise to higher
luminescence than those from IS-Ø-H and Parental-H at all IgG
concentrations. Fig. 5 collectively attested that the IgG produced from
IS-LGD-H was less fucosylated and exhibited better ADCC effects.
Discussion
Improving CHO cell productivity and protein quality is highly desired
for the biopharmaceutical industry, and may be achieved by engineering
the cells to reduce lactate production (28 ), to improve
glycosylation patterns (29 ) or to resist apoptosis (30 ).
To reduce lactate accumulation and IgG fucosylation, we chose to inhibit
LDHA and GFT because LDHA catalyzes lactate production (9 ) while
GFT is essential for fucose-GDT transportation into Golgi for IgG
fucosylation (6 ). In addition, we chose to inhibit DDIT3 because
DDIT3 is implicated in the death of many cells (31 ), but its
roles in CHO cell physiology and recombinant protein production have yet
to be explored. In our preliminary CHO cell culture study, we found that
DDIT3 was upregulated at the late stage of batch culture (Fig. S2). The
DDIT3 upregulation agreed with the notion that ER stress induces DDIT3
expression, which subsequently results in reactive oxygen species (ROS)
response and triggers cell death (10 ). Therefore, we surmised
that inhibiting DDIT3 may promote the longevity of CHO cells during cell
culture.
To suppress these 3 genes, we initially selected CRISPR interference
(CRISPRi) system, which uses deactivated Cas9 (dCas9) fused with a
transcription repressor KRAB (Krüppel-associated box) and the gRNA to
specifically inhibit gene transcription (19 ), because CRISPRi was
recently harnessed for gene repression in CHO cells (32, 33 ).
However, the magnitude of LDHA suppression was less than 40% even
though we screened a large panel of gRNA (Fig. S3), suggesting that
CRISPRi approach may be ineffective in repressing certain endogenous
genes in CHO cells. Since CRISPRi suppresses gene expression by blocking
transcription, whose efficiency may be affected by the local chromatin
structure surrounding the gene (19 ), we therefore exploited a
different mechanism by silencing gene translation using the newly
developed CRISPR-Cas13d system.
We showed that transfection of CRISPR-Cas13d system into CHO cells
effectively knocked down LDHA, GFT and DDIT3, while the efficiency was
highly dependent on the gRNA design (Fig. 1). With the aid of SB
system-mediated integration and Zeocin selection, stable pools of cells
with individual gene knockdown were readily obtained (Fig. 2). The
stable LDHA knockdown alleviated the lactate accumulation (Figs. 2B-2C);
GFT suppression reduced the core fucosylation (Figs. 2D-2E); and DDIT3
knockdown decreased the intracellular DDIT3 protein level (Figs. 2F-2G).
Given these results, we integrated the Cas13d cassette and the gRNA
array co-targeting LDHA, GFT and DDIT3 into the chromosome and achieved
simultaneous knockdown of all 3 genes (Fig. 3A-E), hence establishing a
CHO cell platform for protein production. By integrating the
Herceptin-like IgG gene into the new cell platform,
the engineered cell (IS-LGD-H) conferred significantly higher specific
production rate than the parental CHO cell line (Fig. 3F).
Notably, the negative control IS-Ø-H exhibited faster cell death (Figs.
4A-4B), similar glucose consumption and accumulated more lactate than
the Parental-H cells, probably because simultaneous overexpression of
IgG, Cas13d and the scramble gRNA resulted in aberrantly high ER stress
and metabolic burden to the cells. Nonetheless, IS-Ø-H cells conferred
similar IgG titer when compared with the Parental-H cells. In stark
contrast, by expressing the gRNA array targeting LDHA, GFT and DDIT3,
IS-LGD-H cells exhibited improved longevity (Fig. 4A-4B) and triggered
lower degrees of ROS response (Fig. S4) than the parental cell
(Parental-H) and the negative control (IS-Ø-H). Since DDIT3 induction
triggers subsequent ROS response and cell death (34 ), the
increased tolerance of IS-LGD-H to the adverse culture environment at
the late stage (e.g. at day 10) could be attributable to the efficient
knockdown of DDIT3. Moreover, IS-LGD-H exhibited lower glucose
consumption and lactate accumulation (Figs. 4C-4D), thanks to the
effective silencing of LDHA. The silencing of these two genes allowed
for rewiring of the intrinsic metabolic pathway and enhanced the cell’s
tolerance to environmental stress. As a consequence, the IS-LGD-H
conferred significantly higher IgG titer than Parental-H and IS-Ø-H
(Fig. 4E). Equally importantly, the repression of GFT concomitantly
attenuated the core fucosylation (Fig. 5A) and the resultant IgG
exhibited more potent ADCC efficacy than the IgG produced from the
parental CHO cells (Fig. 5B). These data altogether demonstrated the
feasibility of using CRISPR-Cas13d to engineer CHO cells to improve
antibody production. In addition, one problem in large-scale CHO cell
culture is clumping. We found that the gene encoding clusterin (CLU),
which is implicated in cell aggregation in somatic cells (35 ),
was upregulated along CHO cell culture (Fig. S2). We also exploited
CRISPR-Cas13d to knockdown CLU and hindered CHO cell clumping (Fig. S5),
which further substantiates the potentials of CRISPR-Cas13d for CHO cell
engineering.
CHO cells are commonly engineered by altering the cellular processes,
which can be achieved using different strategies, such as deleting,
silencing or over-expressing individual genes in a cellular pathway.
However, deletion of essential genes such as LDHA is lethal to CHO cells
(36 ). Conversely, gene silencing may be achieved by small
interfering RNA (siRNA) or short hairpin RNA (shRNA). However, siRNA and
shRNA are known to induce serious off-target effects (37, 38 ).
Additionally, high degree of siRNA-mediated knockdown may require
multiple rounds of selection (39 ) as well as additional
phenotypic selection using agents such as LCA (6 ). In comparison,
CRISPR-Cas13d was shown to exhibit more potent knockdown of various
endogenous genes with less off-target effects in multiple cell types
(18, 21, 22, 38, 40 ). The intrinsic ability of Cas13d to process
its pre-crRNA into separate crRNA also allows CRISPR-Cas13d to
simultaneously target multiple mRNA involved in different pathways, thus
enabling concurrent knockdown LDHA, GFT and DDIT3.
In conclusion, we present a novel CRISPR-Cas13d-based approach to
engineering CHO cells in the post-transcriptional level. By efficient
and multiplex knockdown of LDHA, GFT and DDIT3, the engineered CHO cell
accumulates less lactate and is more resistant to cell death, thus
producing more IgG. Furthermore, antibody fucosylation in the engineered
CHO cells is mitigated, thus enhancing the ADCC efficacy. These data
collectively warrant the potentials of CRISPR-Cas13d for CHO cell
engineering and antibody production.
Materials and methods
Design of gRNA spacer
The RNA cleavage efficiency of CRISPR-Cas13 depends on the sequence and
secondary structure of the targeted RNA. We used the RNAfold software
(https://zlab.bio/cas13) to
predict the RNA secondary structure and used siRNA design software,
RNAxs (https://zlab.bio/cas13), to
locate regions with good accessibility of RNA for gRNA spacers design.
For each targeted RNA, we designed and tested 3 gRNA spacers with the
highest scores for gene repression (Fig. S1).
Plasmid construction
The Cas13d gene, CasRx from Ruminococcus flavefaciens strain
XPD3002, and its upstream EF1α promoter were cloned from pXR001
(Addgene) and subcloned into pUSEamp(+) (Merck Millipore) usingPac I/Nhe I. Zeocin resistance gene
(ZeoR)-BGH poly A sequence was amplified from
pSecTag2b (Thermo Fisher) and ligated with a self-cleavable 2A sequence
derived from porcine teschovirus-1 (P2A) to form the
P2A-ZeoR-polyA fragment. This fragment
(P2A-ZeoR-polyA) was sublconed into pUSEAmp(+) to
yield pEF1α-Cas13d-P2A-Zeocin (pCas13d).
Meanwhile, U6 promoter, gRNA backbone and termination sequence were
amplified from pXR003 (Addgene) and subcloned into pTA vector
(Invitrogen) to form a pU6-gRNA vector which expressed the scramble
gRNAØ containing the 30 nt direct repeat without spacer.
A duplex containing 22 bp gRNA spacer for specific gene targeting was
cloned into pU6-gRNA using Bbs I to form a complete pU6-gRNA.
These gRNA cassettes were digested using Mlu I/Hind III and
subcloned into pCas13d to yield different pCas13d-gRNA. For gRNA array
co-targeting LDHA, GFT and DDIT3, we first subcloned the gRNA cassette
targeting GFT into pU6-gRNA that targeted LHDA to yield
pU6-gRNALG. Subsequently, the gRNA cassette targeting
DDIT3 was subcloned into pU6-gRNALG usingBgl II/Hind III to yield pU6-gRNALGD.
To ensure the co-integration of Cas13d and gRNA, we exploited theSleeping Beauty (SB) system, which uses the transposase SB100X to
transpose the gene cassette flanked by IR/DR (inverted repeat/direct
repeat) sequences into the chromosome (24 ). Two IR/DR sequences
and the codon-optimized SB100X gene together with the upstream CMV
promoter were chemically synthesized and cloned into pUSEamp(+) to yield
pIS plasmid. The Cas13d-P2A-ZeoR sequence from
pEF1α-Cas13d-P2A-Zeocin was cloned into pIS usingMlu I/Pvu II to yield pIS-Cas13d. gRNA under U6 promoter
from pU6-gRNAØ or various pU6-gRNA targeting different
genes were cloned into pIS-Cas13d to yield various pIS-gRNA. In
parallel, the gRNA array co-targeting LDHA, GFT and DDIT3 was subcloned
from pU6-gRNALGD into pIS-Cas13d to generate pIS-LGD.
The plasmid ph4D encoding the complete Herceptin-like
anti-HER2 IgG1 was a gift from Dr. Min-Yuan Chou (Industrial Technology
Research Institute, Taiwan).
Cell culture, transfection and selection
CHO-K1 cells (referred to as CHO cells thereafter) were routinely
cultured using F12 medium (#11765, Gibco) containing 10% fetal bovine
serum (FBS). For transient transfection, CHO cells were seeded to
12-well culture plates (1×105 cells/well) and cultured
for 48 h, followed by transfection of 2 µg plasmid DNA using
lipofectamine 3000 transfection reagent (#L3000-015, Invitrogen). At 2
days post-transfection, total RNA was isolated for qRT-PCR assay.
For chromosomal integration into CHO cells, the plasmids (pIS-gRNA,
pIS-LGD or ph4D) were transfected. At day 2, the medium was replaced
with F12 medium containing 10% FBS and 200 µg/ml Zeocin (Invitrogen),
followed by subculture every 4 days. After selection for 7 passages,
stable pools of cells were obtained.
Quantitative real-time reverse transcription PCR (qRT-PCR)
Total RNA was extracted from CHO cells using Quick-RNA Miniprep Kit
(#R1055, ZYMO Research) and reverse transcribed into cDNA using High
Capacity cDNA Reverse Transcription Kit (#4368813, Applied Biosystems).
For quantitative real-time PCR, the reaction was performed with
FastStart Essential DNA Green Master reagents using gene specific
primers (Table S1) on LightCycler96 real-time quantitative PCR system
(Roche Life Science). To analyze the relative expression level of each
target gene, β2-microglobulin (B2M) was used as the internal control
gene and the gene expression levels were normalized to those of control
cells.
Glucose and lactate detection using CuBiAn bioanalyzer
Cell culture supernatant was collected by centrifugation at 1500×g for 5
min and stored at -20°C until analysis. The glucose and lactate
concentrations were analyzed using CuBiAn Biochemical Analyzer (#C125,
Optocell) with a Glucose assay (#200106, 4BioCell) and a Lactate assay
(#200115, 4BioCell).
Lens culinaris agglutinin (LCA) sensitivity assay
Cells were seeded into 96-well plates (1×104cells/well) and cultured at 37°C overnight. The culture medium was
replaced with fresh culture medium containing LCA (Vector Laboratories)
of different concentrations. The cells continued to be cultured at 37°C
for 48 h and the viability was detected using the PrestoBlue assay
(#A13262, Invitrogen) according to user’s manual. The Resazurin in
PrestoBlue can be converted to fluorescent signal in live cells and the
fluorescence was recorded using a 560 nm excitation /590 nm emission
filter set in ELISA reader (SpectraMax® M2, Molecular
Device).
Intracellular immunostaining
Cells were washed with the wash buffer (phosphate-buffered saline (PBS)
containing 0.5% bovine serum albumin (BSA)) and then fixed with 2%
paraformaldehyde at room temperature for 10 min, followed by washing
with the wash buffer. The cells were permeabilized with iced-cold 90%
methanol at room temperature for 10 min and was washed again with the
wash buffer. Cells were immunostained with anti-DDIT3 antibody
(#AP11955b, ABGENT) at a concentration of 0.025 mg/ml for 90 min at
room temperature. After wash, the cells were stained with the secondary
antibody, Goat Anti-Rabbit IgG H&L conjugated with Alexa
Fluor® 488 (#ab150077, Abcam), at a concentration of
0.005 mg/ml. After the staining, DDIT3 expression was analyzed using a
flow cytometer (FACSCalibur, BD Biosciences).
IgG quantification using enzyme-linked immunosorbent assay (ELISA)
For antibody titer analysis by ELISA, 96-well plates were coated with
100 µl/well of recombinant HER2 antigen (2 µg/ml) in 0.1 M
NaHCO3 (pH 9.6) at 4°C for 16 h and washed with PBST
(PBS containing 0.1% Tween 20) three times. The wells in 96-well plates
were blocked by adding StartingBlockTM blocking buffer
(Thermo Scientific) and incubated at 37°C for 1 h, followed by three
PBST washes. The sample or serially diluted standard (anti-HER2 IgG)
were added to the StartingBlockTM buffer and added to
the wells (100 µl/well). After incubation at 37°C for 1 h and three PBST
washes, the secondary antibody (Goat Anti-Human Igκ chain antibody
conjugated with horseradish peroxidase (HRP), Millipore) was diluted
10000 times with StartingBlockTM and added to the
wells. After incubation at 37°C for 1 h, HRP detection was performed
using Tetramethylbenzidine (TMB) (#01016-1, Clinical Science Product)
at room temperature for 15 min. Finally, 2 N HCl (50 µl/well) was added
to stop the reaction and the signal was read at OD450using an ELISA reader.
Specific production rate (SPR) determination
CHO cells were seeded into 6-well plates (1×105cells/well) and cultured with 2 ml growth medium. After 3 days, the cell
numbers were countered and the anti-HER2 IgG titers were determined
using ELISA. The SPR (pg/cell/day) was calculated as described
(41 ).
Adaptation of CHO cells to serum-free medium
After generation of CHO cells capable of producing
Herceptin-like anti-HER2 IgG, the engineered CHO
cells cultured in F12 containing 10% FBS were adapted to chemically
defined serum-free medium (CDM4PERMAb medium, GE Healthcare). Briefly,
the cells were cultured in T-flasks with F12 containing 10% FBS. When
the confluence reached 80%, the medium was replaced with CDM4PERMAb
medium. After 4 days, most cells became detached and the cells were
transferred to the shaker flask (Corning) and cultured at a shaking
speed of 120 rpm.
Production of IgG
After adaption to serum-free medium, the CHO cells expressing the
Herceptin-like anti-HER2 IgG were inoculated into 30
ml of CDM4PERMAb medium containing 4 mM L-glutamine
(3×105 cells/ml). Cells underwent continuous culture
with a shaking speed of 120 rpm at 37°C for 7-10 days. Cell numbers were
countered and glucose and lactate concentrations were monitored every
day. Once the cell viability dropped below 50%, the culture was
terminated.
Antibody-dependent cell-mediated cytotoxicity (ADCC) assay
ADCC effect of IgG was evaluated with an ADCC reporter bioassay
(#G9790, Promega). The assay uses hFcγRⅢa+ Jurkat T
cell that expresses a luciferase reporter driven by an NFAT-response
element as the effector cell and HER2-rich SKBR3 breast cancer cell as
the target cell. The HER2 receptor on SKBR3 is recognized by the
Herceptin-like IgG whose Fc region binds the FcγRⅢa
receptor and triggers the luciferase expression in the effector cells.
The IgG was harvested when the viability dropped below or near 50% and
was first purified using Mabselect SuRe® resin
(#17-5474-021, GE Healthcare) following the manufacturer’s
instructions. In brief, the culture supernatant was collected,
centrifuged (4500×g, 10 min) and loaded to a column packed with 0.2 ml
Mabselect SuRe® resin. After washing with Dulbecco’s
PBS and elution with Arginine-HCl (25 mM, pH 2.5), the purified IgG was
neutralized with minute amount of Tris-HCl (1 M) and stored at 4°C until
analysis.
For the ADCC reporter assay, the purified IgG was 10-fold serially
diluted using RPMI-1640 medium containing 0.5% FBS (low IgG). SKBR3
cells (100 µl) were seeded to each well of 96 well plates
(1×104 cells/well) and cultured overnight. After
removal of supernatant, we added the serially diluted IgG and added 25
µl Jurkat-hFcγRⅢa-NFAT effector cells (1.5×105cells/well). After 6 h of reaction at 37°C, 75 µl Bio-Glo™ Luciferase
Assay Buffer was added to detect the luciferase activity in the effector
cells. The luciferase activity was read using GloMax®Navigator Microplate Luminometer for the quantification of ADCC effect
of IgG.
Acknowledgement
The authors acknowledge the financial support from the Ministry of
Science and Technology (MOST 107-2622-E-007-003-CC1,
108-2622-E-007-012-CC1, 109-2634-F-007-023). This work was also
financially supported by the Frontier Research Center on Fundamental and
Applied Sciences of Matters, from The Featured Areas Research Center
Program within the framework of the Higher Education Sprout Project by
the Ministry of Education (MOE 109QR001I5), Taiwan.
Conflicts of Interests
The authors declare no conflicts of interests.
Supporting Information : Fig. S1, Fig. S2, Fig. S3, Fig. S4.
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Figure Captions
Fig. 1. Design of CRISPR-Cas13d for gene knockdown in CHO
cells. (A) Schematic illustration of pCas13d-gRNA that expressed Cas13d
under EF-1α promoter and gRNA under U6 promoter. (B) Sequences of the
gRNA composed of 5’ direct repeat and 3’ spacer to target the
complementary mRNA sequence of LDHA, GFT and DDIT3. (C) Targeting
positions on the coding sequences of LDHA, GFT and DDIT3. (D) Relative
mRNA levels of LDHA. (E) Relative mRNA levels of GFT. (D) Relative mRNA
levels of DDIT3. We designed 3 gRNA for LDHA and DDIT3 but only 1 gRNA
for GFT because the predicted spacers were in very close proximity (Fig.
S1). We constructed a set of pCas13d-gRNA expressing individual gRNA and
transfected the plasmid into CHO-K1 (referred to as CHO thereafter)
cells. Gene expression was analyzed at 2 days post-transfection and
normalized to that in the cells transfected with the plasmid expressing
the scramble gRNAØ.
Fig. 2. Chromosomal integration of CRISPR-Cas13d aided bySleeping Beauty (SB) system enabled individual gene knockdown and
altered CHO cell phenotype. (A) Schematic illustration of pIS-gRNA
plasmids that incorporated the SB system and CRISPR-Cas13d. The plasmid
expressed SB100X, ZeoR, Cas13d and the gRNA targeting
either LDHA, GFT or DDIT3. The CRISPR-Cas13d module and
ZeoR were flanked by a pair of IR/DR sequences for
SB100X recognition. (B) Relative LDHA mRNA level. (C) Lactate
production. (D) Relative GFT mRNA level. (E) LCA assay. (F) Relative
DDIT3 mRNA level. (G) Intracellular DDIT3 protein levels. The cells were
transfected with different pIS-gRNA (including pIS-Ø) plasmids and
selected with Zeocin to yield stable pools of cells. The mRNA expression
levels were analyzed by qRT-PCR and normalized to those of the cells
transfected with pIS-Ø. The cells were subjected to lactate assay and
LCA assay at day 2 after subculture. The DDIT3 protein levels were
analyzed at day 2 and 6 by intracellular immunostaining and analyzed by
flow cytometry.
Fig. 3. Establishing a stable CHO cell platform with
simultaneous knockdown of LDHA, GFT and DDIT3. (A) Illustration of
pIS-LGD plasmid that was similar to pIS-gRNA except that the gRNA was an
array co-targeting LDHA, GFT or DDIT3. (B) Relative Cas13d mRNA level.
(C) Relative LDHA mRNA levels. (D) Relative GFT mRNA levels. (E)
Relative DDIT3 mRNA levels. (F) Specific production rate (SPR). The CHO
cells were transfected with pIS-LGD and selected with Zeocin to yield
stable pools of cells (IS-LGD). The gene expression levels were analyzed
by qRT-PCR and normalized to the control transfected with pIS-Ø (IS-Ø).
The SPR was calculated by measuring the IgG titer and cell number for 3
days.
Fig. 4. CRISPR-Cas13d-mediated knockdown improved the cell
phenotype and IgG production. (A) Viable cell density (VCD). (B)
Viability. (C) Glucose concentration. (D) Lactate concentration. (E) IgG
titer. The cells were adapted to serum-free medium and cultured in
shaker flasks in a batch mode. The VCD, viability, glucose and lactate
were measured daily until the viability dropped below or near 50%. The
IgG titer was determined at the end of culture by ELISA.
Fig. 5. Quality analysis of IgG produced the engineered cells.
(A) LCA assay. (B) ADCC reporter assay.