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Eng. Life Sci. 2015,15, 660–666 www.els-journal.com
Tao Sun1,2
Chaodong Li1,2
Lei Han1,2
Hua Jiang3
Yueqing Xie3
Baohong Zhang1,2
Xiuping Qian1,2
Huili Lu1,2
Jianwei Zhu1,2, 3
1School of Pharmacy, Shanghai
Jiao Tong University, Shanghai,
China
2Engineering Research Center of
Cell & Therapeutic Antibody,
Ministry Of Education (MOE),
Shanghai, China
3Jecho Laboratories, Inc.,
Frederick, MD, USA
Short Communication
Functional knockout of FUT8 in Chinese
hamster ovary cells using CRISPR/Cas9 to
produce a defucosylated antibody
We report the adaptation of the new CRISPR/Cas9 (clustered regularly interspaced
short palindromic repeat/CRISPR-associated protein 9) system to disrupt the gene
encoding fucosyltransferase 8 (FUT8), an α1,6-fucosyltransferase that directs fu-
cose addition to derived antibody Fc region asparagine 297, in Chinese hamster
ovary (CHO) cells. Compared to previously reported homologous recombination
or zinc-finger nucleases (ZFNs) applications in CHO cells, CRISPR/Cas9 demon-
strated higher targeting efficiency and easier customization. FUT8 disruptive clones
(FUT8−/−) were obtained within 3 weeks at indel frequencies ranging from 9 to
25%, which could be enhanced to 52% with Lens culinaris agglutinin (LCA) selec-
tion. Based on the lectin blot method, the derived FUT8−/−clonehadtheabilityto
produce defucosylated therapeutic mAb with no detrimental effects on cell growth,
viability, or product quality. The clone had the potential of industrial application for
therapeutic antibodies manufacturing. We have demonstrated functionally that a
gene related to product synthesis could be mutated using CRISPR/Cas9 technology,
and consequently the glycan profile of expressed mAb was alternated. We believe
that with its robustness and effectiveness, CRISPR/Cas9 can be widely applicable in
cell line development leading to higher productivity and better quality of mAbs and
other biological therapeutics.
Keywords: Antibody / Antibody-dependent cellular cytotoxicity / CHO-K1 cells / CRISPR/Cas9
/ Fucose
Additional supporting information may be found in the online version of this article at
the publisher’s web-site
Received: October 23, 2014; revised: May 29, 2015; accepted: June 15, 2015
DOI: 10.1002/elsc.201400218
1 Introduction
mAb therapeutics is increasing rapidly in treatment of various
diseases [1]. There are more than 30 antibody therapeutics ap-
proved by regulatory agencies for the worldwide market, and
many more mAb candidates are in clinical trials [2]. Antibod-
ies execute their function by destroying targets through two
Correspondence: Jianwei Zhu (jianweiz@sjtu.edu.cn), School of
Pharmacy, Shanghai Jiao Tong University, Minhang 200240, Shang-
hai, China
Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CHO,
Chinese hamster ovary; CRISPR/Cas9, clustered regularly interspaced
short palindromic repeat/CRISPR-associated protein 9; FUT8,fucosyl-
transferase 8 (α1,6-fucosyltransferase); LCA,Lens culinaris agglutinin;
sgRNA, single guide RNA; TALEN , transcription activator-like effector
nuclease; ZFN, zinc-finger nuclease
pathways: complement-dependent cytotoxicity and antibody-
dependent cellular cytotoxicity (ADCC) [3]. ADCC is mediated
by antibody Fc region binding to lymphocyte receptors [4]. N-
oligosaccharide is present on asparagine 297 of IgG heavy chain
Fc region, whereas fucose is commonly found in the oligosac-
charide structure, which could hinder Fc binding to lymphocyte
receptors [5]. Compared to fucosylated antibodies derived from
wild-type Chinese hamster ovary (CHO) cells, defucosylated
antibodies achieved 100-fold higher ADCC activity in vitro [6].
Therefore, antibody defucosylation is considered to be a power-
ful approach to enhance ADCC activity.
The majority of commercial antibodies are produced from
CHO cells, with advantages of posttranslational modifications,
Additional correspondence: Huili Lu (roadeer@sjtu.edu.cn), School of
Pharmacy, Shanghai Jiao Tong University, Minhang 200240, Shanghai
China
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such as glycosylation, phosphorylation, acetylation, etc [7].
However, it is notable that core fucosylation commonly found
in mAbs derived from CHO cells would hinder antibodies bind-
ing to their receptors [8]. In CHO cells, fucosyltransferase 8
(FUT8) is an α1,6-fucosyltransferase that directs fucose addition
to asparagine-linked N-acetylglucosamine moieties, a common
feature of N-linked glycan core structures [9]. So far, the effect of
disrupting FUT8 gene to enhance ADCC has been reported by
several groups using different approaches, such as homologous
recombination [10] or zinc-finger nucleases (ZFNs) [11], both
achieved increased ADCC activity (approximately 100-fold).
Traditionally, homologous recombination was used for
genome editing in cell lines and animal models, though it was
less efficient and was a time-consuming process due to technical
complexity [12]. This was particularly true in the case of FUT8
disruption, as approximately 120 000 clones were screened to
obtain only three FUT8−/−clones with a normal growth pro-
file [13]. To improve the efficiency of disrupting a coding gene,
technologies such as ZFNs and transcription activator-like ef-
fector nucleases (TALENs) were developed and reported [14].
Compared to traditional homologous recombination, ZFNs and
TALENs demonstrated higher genome editing efficiency [15].
These nucleases could introduce double-strand breaks, which
would be repaired by one of the following two major mecha-
nisms: the error-prone nonhomologous end-joining or the high-
fidelity homology-directed repair pathway [16]. Nonhomolo-
gous end-joining can be harnessed to mediate gene functional
disruptions, as indels occurring in a coding region could lead to
frameshift mutations and protein function loss [17]. ZFNs and
TALENs were extremely expensive and difficult to design, which
limited their widespread use. More recently, CRISPR (clustered
regularly interspaced short palindromic repeat)/Cas9 (CRISPR-
associated protein 9) system, which was derived from the mi-
crobial adaptive immune system [18], has been developed as an
efficient genome editing tool and applied to diverse species such
as plants, animals, bacteria, and yeast [19], as well as in CHO
cells [20]. This system required only Cas9 and single guide RNA
(sgRNA) [21], giving it several advantages including ease of cus-
tomization, higher targeting efficiency, and ability to facilitate
multiplex genome editing [22].
Here,wereportedanewproceduretodisruptFUT8
gene in CHO-K1 cell line using CRISPR/Cas9 system. Lens
culinaris agglutinin (LCA) based phenotypic screen was used
to enrich clones with exclusively doubly modified alleles. mAbs
produced in the CHO FUT8−/−cells were defucosylated.Further,
we demonstrated that the gene modification had no detrimental
effects on cell growth, viability, or product quality.
2 Materials and methods
2.1 Construction of FUT8 disruptive vector
The exon 10 of FUT8 (GeneBank ID: 100751648) was selected
as the target site for mutagenesis because it encoded for the cat-
alytic site of the enzyme. Five sgRNAs were designed, based
on previously described design rules [19] to obtain satisfied
indel frequencies (Supporting information Tables S1–S3 and
Supporting information, Fig. S1). Exon 10 and adjacent re-
gion (685 bp) was amplified by PCR using primers FUT8-F
(5’-CTGTTGATTCCAGGTTCCCATATA-3) and FUT8-R (5-
TTGA ATGATGACTGCTA GTGATGCT -3). The plasmid pX330
was kindly provided by Dr. Zhang Feng (MIT) [19] and digested
with BbsI (New England Biolabs, MA, USA); a pair of annealed
sgRNA was ligated to the linear plasmid. All cloning steps were
confirmed by DNA sequencing at Invitrogen (Shanghai, China),
derived plasmids were addressed as pX330-sgRNA15.
2.2 Analysis of Cas9 activity in CHO-K1 cells
Cells were incubated at 37◦C with 5% CO2and transfected using
Lipofectamine 2000 reagent (Invitrogen, CA, USA). Forty-eight
hours posttransfection cells were harvested and genome DNA
was extracted using Genome DNA Extraction Kit (Axygen, CA,
USA). Approximately 200 ng PCR product of FUT8 exon 10 re-
gion (685 bp) was annealed using the following program: heated
to 95◦C for 3 min and then ramped down to 25◦Cat5
◦C/min.
Annealed PCR fragment was treated with 0.3 μL T7 endonucle-
ase at 37◦C for 45 min, followed by analysis on 1% agarose gel.
Related indels were stained with ethidium bromide and detected
under gel imaging systems (Tanon, Shanghai, China). Percent-
age of indel frequency was calculated using ImageJ software as
previously described by Hwang et al. [23].
2.3 Generation of CHO FUT8−/−clones
Cells were exposed to 100 μg/mL LCA (Vector Labs, CA, USA)
2 days posttransfection. After 6-day growth in the presence of
100 μg/mL LCA, clones morphologically similar to the wild-type
CHO-K1 were expanded. Limiting dilution was performed to
screen doubly modified alleles’ clones. When clones reached an
80% confluence, genome DNA extraction and T7 endonuclease
digestion were performed as described above. To identify both
alleles’ modification in LCA-resistant clones, PCR product from
each T7E1-positive clone was TA-cloned using TA cloning kit
(Takara, Tokyo, Japan). At least 10 colonies were picked from
each transformation for DNA sequencing as CHO cells were
aneuploids.
2.4 LCA reactivity and genetic stability analysis
FITC-LCA binding assay was performed to test FUT8−/−clones
phenotypic profiles. Briefly described, cells were seeded in 12-
well plates and incubated in the presence of 20 μg/mL FITC-
LCA (Vector Labs) for 30 min. Cells were washed three times
and visualized under fluorescence microscope (Olympus, Tokyo,
Japan). An LCA-binding test based on flow cytometer analysis
was also performed as previously described [13]. Clones were
cultured for 10 passages to confirm the genetic stability of the
modified clones, both alleles’ DNA sequencing was performed
as described above.
2.5 Anti-CD20 mAb expression and purification
Chimeric anti-CD20 mAb (Rituximab, drug bank: DB00073)
was chosen as a model antibody [24]. The heavy chain and light
chain were separately subcloned into pcDNA3.1 (Invitrogen)
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as expression vectors (heavy chain and light chain sequences
are shown in Supporting information). All cloning steps were
confirmed by DNA sequencing. Stable transfected cell pools of
CHO-K1 cells and FUT8−/−clones were selected via 800 μg/mL
G418 pressure for 10 days. Serum-free adaptationwas performed
to eliminate the influence of FBS presence. Cell count and via-
bility were determined by Trypan blue method. AKTA purifier
system (GE Healthcare, CT, USA) was used to purify antibodies
from culture supernatants as previously described [25].
2.6 SDS-PAGE, western blot, and lectin blot
Protein fractions from protein A chromatograph were analyzed
by 10% SDS-PAGE to confirm that purified glycoproteins were
antibodies. Protein loading was normalized to 500 ng/well for
SDS-PAGE, 50 ng/well for lectin blot, and 5 ng/well for western
blot. For lectin blot, the membrane was incubated with 10 μg/mL
FITC-LCA for 30 min in the dark, and the green fluorescence
signal was scanned by multiple function laser scanner (Typhoon
FLA 9500, GE Healthcare).
3 Results
3.1 FUT8 modification generated by CRISPR/Cas9
The exon 10 encoded for the catalytic site inverting substrate
GDP-fucose, therefore was chosen as the target for CRISPR/Cas9
editing. Plasmid containing a chimeric guide RNA structure
had higher genome editing efficiency as compared to plasmid
containing a crRNA-tracrRNA guide RNA [19], hence plasmid
pX330 containing human U6 polymerase promoter generat-
ing chimeric sgRNA was selected to perform gene perturba-
tion, which was confirmed to be effective in multiple species
as well as Chinese hamster [20]. Two days posttransfection, T7
endonuclease digestion tests were performed in cells transfected
with diverse pX330-sgRNAs. Indels were stained with ethidium
bromide and visualized under DNA gel imaging system, indel
frequencies (%) were analyzed using ImageJ software with the
value ranging from 9 to 24% as indicated in Fig. 1A. No indels
were visualized without pX330-sgRNA treatment.
3.2 FUT8 functional disruptive clones generated by
limiting dilution
To produce defucosylation mAbs in CRISPR/Cas9 derived
clones, both alleles of FUT8 gene should be disrupted. LCA is a
plant lectin with high specificity to bind IgG N-glycan core fu-
cose, which can lead to cell death [26]. CHO FUT8−/−clones are
able to survive in the presence of LCA as cells fail to synthesize fu-
cosylated glycoproteins. Inour experiment, cells were transferred
to 12-well plates 2 days posttransfection and LCA was added to
each well at a final concentration of 100 μg/mL (day 2). Eighteen
hours later, most cells became round and got detached from the
plate, the phenotype similar to wild-type clones appeared 2 days
after LCA treatment (day 4, Fig. 1B). Cells were LCA selected for
6 days and expanded after fresh medium replacement (day 8).
As shown in Fig. 1A, indel frequencies of LCA-resistant clones
were ranging from 24 to 52%, indicating that LCA treatment en-
riched FUT8−/−clones. No LCA-resistant clones were detected
in the absence of pX330-sgRNA transfection within 6 days LCA
exposure.
LCA-selected clones derived from pX330-sgRNA1 transfec-
tion were subcloned by limiting dilution. Three clones, named
D5, C9, and C10 showing positive T7E1 digestion activity were
chosen for further research. Exon 10 region was amplified by
PCR from these clones, both alleles’ genotypes of clone D5, C9,
and C10 are listed in Fig. 1C. These modifications were consid-
ered to lead FUT8 truncation and frameshift mutations in these
clones. C9 clone was morphologically different from wild-type
CHO cells (Supporting information, Fig. S2), which indicated
that other alternations in cell metabolism might occur besides
the FUT8 disruption. C10 clone was successfully adapted to
serum-free medium and had the best cell growth among the
three clones, therefore was selected for further studies.
There was no available commercial antibody that could dis-
tinguish FUT8 exon 10 functional disruption. We therefore tested
the FUT8 activity by LCA-resistance phenotype method. It was
confirmed by fluorescence microscopy images and flow cytome-
ter analysis that these clones lacked the ability to bind FITC-LCA.
As indicated in Fig. 2A and B and Supporting information Fig. S3,
FITC-LCA could bind to wild-type CHO-K1 cells but not the
FUT8−/−clones. Genetic stability of C10 clone was also evalu-
ated for 10 passages. No alternation was found in genotype or
LCA-resistance ability compared to the parental clone (data not
shown).
3.3 Antibodies expression and lectin blot based
fucosylation detection
Prior to express an antibody in the cells, growth profiles of C10
clone and wild-type CHO-K1 cells were compared side by side
in 125-mL shaker flasks with 20 mL working-volume medium.
During 8 days incubation, both CHO-K1 cells and C10 clone
cell densities reached greater than 6 ×106cells/mL and main-
tained viability above 90%. As shown in Fig. 2C and D, C10
clone had comparable viable cell counts and culture viability
compared to wild-type CHO-K1 cells throughout three parallel
experiments.
Cell pools stably expressing anti-CD20 antibody were derived
from both wild-type CHO-K1 cells and C10 clone using G418 se-
lection. Each cell pool was cultured in a 1000-mL shake flask con-
taining 300 mL CD-CHO medium supplemented with HT and
glutamine, supernatants were collected when cell viabilities were
below 30%. Proteins of interest in the supernatants were purified
by AKTA purifier system and analyzed by SDS-PAGE and western
blot (Fig. 3A and B). Roughly equivalent quantity of purified IgGs
were obtained from C10 clone and CHO-K1 cells pools under
the same cell incubation condition based on bicinchoninic acid
assays, with expression levels around 1.2 mg/L. FITC-LCA lectin
blot was chosen as an alternative method to examine whether
antibody derived from the C10 clone contained fucose [27].
As expected, antibody derived from C10 clone showed no visi-
ble fluorescence compared to antibody from parental CHO-K1
cells under both nonreducing and reducing conditions in three
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Figure 1. Genome editing in CHO-K1 cells by CRISPR/Cas9. (A) T7E1 digestion for Cas9 activity in CHO cells. Two hundred nanograms
PCR product was digested and loaded for each lane. Left panel, genomic DNA was extracted from CHO-K1 cells 2 days after transfected
with different pX330-sgRNAs. Right panel, genomic DNA was extracted from expanded clones by LCA selection. Note that indel frequency
was not completely equivalent to disruption efficiency. (B) Wild-type CHO-K1 cells were round and detached from the plate when treated
with 100 μg/mL LCA for 18 h, while pX330-sgRNAs transfected clones with wild-type morphology appeared under the same condition. LCA
precipitate was visible. (C) Sanger sequences of FUT8 exon 10 mutated alleles obtained from three CHO FUT8−/−clones. Alleles were
designated as A and B. The gap shown in the reference sequences has been included for better representation of alignment results. All
modifications could lead to FUT8 frameshift mutations.
parallel experiments (Fig. 3C). Weconcluded that CHO FUT8−/−
clone generated by CRISPR/Cas9 was able to express mAb with
significantly reduced fucosylation.
4 Discussion
In this report, we presented successful FUT8-functional disrup-
tion in CHO-K1 cells using CRISPR/Cas9 system. Normally, the
20–30 bp region downstream of the start codon ATG was chosen
as the target [28], while catalytic site of FUT8 protein was chosen
for successful gene perturbation in CHO-K1 cells as previously
reported [8]. Off-target mutagenesis was always an undesirable
effect induced by Cas9 and hard to avoid, enhancement of Cas9
targeting specificity seemed to be promising as many researchers
had fruitful achievements of improved Cas9 target recognition
fidelity [29, 30]. A successful gene disruption in COSMC and
FUT8 with high indel frequency was reported, in addition to
gene disruption results that showed consistency as reported by
Ronda et al. [20], we were able to demonstrate the final product
without fucose.
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Figure 2. LCA reactivity and cell growth of CHO FUT8−/−C10 clone. (A) FITC-LCA failed to bind C10 clone membrane glycoproteins. Under
panels, cells were treated with 20 μg/mL FITC-LCA, fluorescence microscopy images were subsequently acquired at a green fluorescent
protein channel. (B) FACS paragraphs of FITC-LCA stained cells. x-axis, fluorescence intensity; y-axis, cell counts. NC refers to negative
control wild-type CHO-K1 cells without FITC-LCA treatment. For details see Supporting information Fig. S3. Cell growth (C) and viability (D)
of serum-free adapted C10 clone and CHO-K1 cells. Cell density and cell viability were examined in 125-mL shaker flasks (20 mL working
volume) during 8 days incubation. Error bars represent the SD from three parallel experiments.
LCA selection was conducted to obtain stable FUT8−/−clones
within a short duration. Another gene related to core fucose
synthesis pathway named GDP-fucose transporter was also dis-
rupted in our lab (data not shown), which was responsible for
transporting GDP-fucose into Golgi apparatus [31]. GDP-fucose
transporter modified clones showed no resistance to LCA, indi-
cated that other GDP-fucose transporting pathways could play
complementary roles.
A new host cell line to produce completely defucosylated an-
tibodies, needs to be characterized in terms of growth, viability,
and antibody productivity [32]. In this study, we compared the
C10 clone and wild-type CHO-K1 cells in a shake flask sys-
tem. Cell cultures were maintained for 8 days before significant
clumping occurred. For further comparison, feb-batch cultiva-
tion in a bioreactor system would be preferable. Our data showed
CHO FUT8−/−C10 clone had comparable viable cell counts and
viability throughout three parallel experiments compared to the
wild-type cell line.
To detect the presence of fucose in the mAb glycan structure,
we employed a simple and quick FITC-LCA lectin blot method,
whereas other approaches such as MALDI-TOLF-MS and high
pH anion exchange chromatography with pulsed amperometric
detection could be utilized [33]. FITC-LCA showed very high
specificity binding to α1,6-fucose and was a widely accepted
approach to confirm N-glycan fucosylation of glycoproteins [27,
34]. As expected, antibody produced in C10 clone showed no
detectable fluorescence compared to antibody from the parental
CHO-K1 cells under both nonreducing and reducing conditions,
indicating that the C10 clone produced defucosylated antibodies.
In summary, our derived CHO FUT8−/−clone had the ability
to produce defucosylated antibody with no detrimental effects
in cell growth, viability, and the potential for industrial utiliza-
tion. We demonstrated CRISPR/Cas9 application in CHO-K1
cells genome editing as well as its effect on product glycosyla-
tion alternation, which would be potentially useful in indus-
trial purposes [35, 36]. To develop the potential of CRISPR/Cas9
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Figure 3. Biochemical analysis of IgG purified from C10 clone and CHO-K1 cells transfected with anti-CD20 mAb expression vectors. (A)
SDS-PAGE and Coomassie brilliant blue staining of the purified IgG. (B) Western blot of the purified IgG. Anti-CD20 mAbs were detected
by HRP-labeled goat antihuman Fc antibody and ECL. (C) The PVDF membrane was incubated with 10 μg/mL FITC-LCA, and subsequently
detected using multifunctional laser scanner after washing three times under a 488 nm channel. Note that proteins were normalized to
500 ng/well for SDS-PAGE, 50 ng/well for lectin blot, and 5 ng/well for western blot.
application in bioprocess, other genes related to recombinant
protein productivity or quality enhancement are currently be-
ing investigated in our labs. We believe that as a robust genome
editing tool, CRISPR/Cas9 system can be widely applicable in
cell line development for manufacturing high-quality mAbs or
other biological therapeutics.
We thank F. Zhang of the Broad Institute of MIT and Harvard for
kindly providing us with the Cas9 expression vector. The research
was supported by Chinese Natural Science Foundation #81473127.
The authors have declared no conflict of interest.
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