Establishment and Application of Multigene Engineering Transformation Strategy for CHO Cells

doi:10.13560/j.cnki.biotech.bull.1985.2022-0863

Abstract

Abstract:

In recent years, CHO（Chinese hamster ovary）cell engineering transformation mainly changes a single function of cells by knock-in or knock-out genes, but the knock-in and knock-out genes often cannot simultaneously play the corresponding functions in a single experimental operation, which limits the application of multigene synchronous transformation. In this study, Bcl-2, the anti-apoptotic gene in the apoptosis pathway, and FUT8, the key enzyme gene of fucose synthesis in the protein fucosylation pathway, were selected as the knock-in and knock-out genes. CRISPR/Cas9 technology was used to establish the site-specific synchronous knock-in and knock-out gene editing strategy. The monoclonal cell line obtained by using this strategy overexpressed Bcl-2 protein and lost FUT8 protease function. After subculture, it was found that the gene expressions of the cell lines obtained by the established site-specific synchronous knock-in and knock-out gene editing strategy were stable within 60 d. Compared with the original wild-type cells, the cell line demonstrated higher tolerance to serum deprivation and stronger resistance to death. This reveals that the gene editing strategy based on site-specific synchronous knock-in and knock-out is feasible and can be used to construct CHO engineering cell lines for recombinant protein production.

Key words: CHO cells, CRISPR/Cas9, gene editing, site-specific synchronization, cell line construction

CHENG Jing-wen, CAO Lei, ZHANG Yan-min, YE Qian, CHEN Min, TAN Wen-song, ZHAO Liang. Establishment and Application of Multigene Engineering Transformation Strategy for CHO Cells[J]. Biotechnology Bulletin, 2023, 39(2): 283-291.

Figures/Tables 12

Table 1 sgRNA oligos

寡核苷酸链名称Oligos name	DNA序列DNA sequences（5'-3'）
FUT8-sg1F	CACCGATGGACTGGTTCCTGGCGT
FUT8-sg1 R	AAACACGCCAGGAACCAGTCCATC
FUT8-sg2 F	CACCGTAAAACAATAAGGTCCCCC
FUT8-sg2 R	AAACGGGGGACCTTATTGTTTTAC
FUT8-sg3 F	CACCGAGAAGGCCCTATTGATCAG
FUT8-sg3 R	AAACCTGATCAATAGGGCCTTCTC

Table 2 PCR primers for T7E I enzymatic digestion assay

引物名称Primer name	DNA序列DNA sequence（5'-3'）
FUT8-Exon1-F	AGAGTCATCACAGTATACCAGAGAG
FUT8-Exon1-R	GCCACTGCTTCTATATACTGATTCA
FUT8-Exon2-F	CATTCTCAGCTAGCCCTTATGATTA
FUT8-Exon2-R	TATGGAAGCCCAAATGAAGCACA

Fig. 1 Map of donor plasmid

Table 3 Primers for site-specific integration

目的 Purpose	引物名称 Primer name	DNA序列 DNA sequence（5'-3'）
5' junction PCR	FUT8 5' F	AACTCTGATTTTTGGAATCCCCTTTCTTCAGC
5' junction PCR	FUT8 5' R	TGGGTCTCCCTATAGTGAGTCGTATTAATTTCG
3' junction PCR	FUT8 3' F	ATGAAGCAGCACGACTTCTTCAAGTCC
3' junction PCR	FUT8 3' R	GCAATGGATGCAAACAGTGGTGTGG
Out-out PCR	FUT8 5' F	AACTCTGATTTTTGGAATCCCCTTTCTTCAGC
Out-out PCR	FUT8 3' R	GCAATGGATGCAAACAGTGGTGTGG

Table 4 Primers for qPCR

引物名称Primer name	DNA序列DNA sequence（5'-3'）
M-Bcl2 F	TCACAGAAGGACAAGGTGGATT
M-Bcl2 R	AATGCTGACCTGAGCTGGTTT
H-Bcl2 F	GAACTGGGGGAGGATTGTGG
H-Bcl2 R	CATCCCAGCCTCCGTTATCC
GAPDH F	CATGGCCTTCCGTGTTCCTA
GAPDH R	TGAAGTCGCAGGAGACAACC
FUT8 F	GACCACCCTGACCATTCTAGC
FUT8 R	CACGGACTCTTCCTGTAGCTG

Fig. 2 Results of sgRNA gene editing efficiency M: DL2000 DNA marker. 1: Wild-type cells exon1 PCR amplification products. 2: Wild-type cells exon 2 PCR amplification products. 3: PCR amplification products of sgRNA1/Cas9 transfected cells. 4: Exon1-sgRNA2/Cas9 transfected cells PCR amplification products. 5: PCR amplification products of sgRNA3/Cas9 transfected cells

Fig. 3 Schematic diagram of the site-specific synchronous knock-in and knock-out gene editing strategy

Fig. 4 Molecular identification results of the site-specific synchronous double-knock cell A: Electrophoresis results of 5' junction PCR. B: Electrophoresis results of 5' junction PCR. C: Electrophoresis results of out-out PCR. D: Sequencing results of 5'/3' junction PCR amplification products. M: DL5000 DNA marker. 1: Wildtype CHO-K1 cells. 2: Site-specific synchronous double-knock cell

Fig. 5 Gene expressions of the site-specific synchronous double-knock cell A: Relative mRNA expression of endogenous Bcl-2. B: Relative mRNA expression of exogenous Bcl-2. C: Relative mRNA expression of FUT8. D: EGFP protein expression. E: Bcl-2 protein expression. F: FUT8 protease function. ** P<0.01，**** P<0.000 1，n=3. The same below

Fig. 6 Stability results of gene expression A: Proportion of EGFP protein expression in cells. B: Fluorescence mean intensity of cells

Fig. 7 Cell viability under serum-free condition Left part: Cell viability at 72 h. Right part: Cell viability at 96 h. * P<0.05，n=3. The same below

Fig. 8 Evaluation on the anti-apoptotic properties of the site-specific synchronous double-knock cell A: Proportion of necrotic cells. B: Proportion of early apoptotic. C: Proportion of late apoptotic. D: Proportion of living cells. n=3

References 26

[1]	Lin PC, Chan KF, Kiess IA, et al. Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologics[J]. mAbs, 2019, 11(5): 965-976. doi: 10.1080/19420862.2019.1612690 URL
[2]	Hacker DL, Balasubramanian S. Recombinant protein production from stable mammalian cell lines and pools[J]. Curr Opin Struct Biol, 2016, 38: 129-136. doi: 10.1016/j.sbi.2016.06.005 URL
[3]	Dahodwala H, Lee KH. The fickle CHO: a review of the causes, implications, and potential alleviation of the CHO cell line instability problem[J]. Curr Opin Biotechnol, 2019, 60: 128-137. doi: 10.1016/j.copbio.2019.01.011 URL
[4]	Ha TK, Kim D, Kim CL, et al. Factors affecting the quality of therapeutic proteins in recombinant Chinese hamster ovary cell culture[J]. Biotechnol Adv, 2022, 54: 107831. doi: 10.1016/j.biotechadv.2021.107831 URL
[5]	Hartley F, Walker T, Chung V, et al. Mechanisms driving the lactate switch in Chinese hamster ovary cells[J]. Biotechnol Bioeng, 2018, 115(8): 1890-1903. doi: 10.1002/bit.26603 pmid: 29603726
[6]	Pereira S, Kildegaard HF, Andersen MR. Impact of CHO metabolism on cell growth and protein production: an overview of toxic and inhibiting metabolites and nutrients[J]. Biotechnol J, 2018, 13(3): e1700499.
[7]	Xu X, Nagarajan H, Lewis NE, et al. The genomic sequence of the Chinese hamster ovary(CHO)-K1 cell line[J]. Nat Biotechnol, 2011, 29(8): 735-741. doi: 10.1038/nbt.1932 URL
[8]	Zhou H, Liu ZG, Sun ZW, et al. Generation of stable cell lines by site-specific integration of transgenes into engineered Chinese hamster ovary strains using an FLP-FRT system[J]. J Biotechnol, 2010, 147(2): 122-129. doi: 10.1016/j.jbiotec.2010.03.020 pmid: 20371256
[9]	Fischer S, Handrick R, Otte K. The art of CHO cell engineering: a comprehensive retrospect and future perspectives[J]. Biotechnol Adv, 2015, 33(8): 1878-1896. doi: 10.1016/j.biotechadv.2015.10.015 pmid: 26523782
[10]	Lee JS, Grav LM, Lewis NE, et al. CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives[J]. Biotechnol J, 2015, 10(7): 979-994. doi: 10.1002/biot.201500082 pmid: 26058577
[11]	Tang DM, Subramanian J, Haley B, et al. Pyruvate kinase muscle-1 expression appears to drive lactogenic behavior in CHO cell lines, triggering lower viability and productivity: a case study[J]. Biotechnol J, 2019, 14(4): e1800332.
[12]	Ha TK, Hansen AH, Kildegaard HF, et al. Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures[J]. Metab Eng, 2020, 57: 182-192. doi: S1096-7176(19)30242-3 pmid: 31785386
[13]	Misaki R, Iwasaki M, Takechi H, et al. Establishment of serum-free adapted Chinese hamster ovary cells with double knockout of GDP-mannose-4, 6-dehydratase and GDP-fucose transporter[J]. Cytotechnology, 2022, 74(1): 163-179. doi: 10.1007/s10616-021-00501-3 pmid: 35185292
[14]	Safari F, Farajnia S, Ghasemi Y, et al. Multiplex genome editing in Chinese hamster ovary cell line using all-in-one and HITI CRISPR technology[J]. Adv Pharm Bull, 2021, 11(2): 343-350. doi: 10.34172/apb.2021.032 pmid: 33880357
[15]	Tan JGL, Lee YY, Wang TH, et al. Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors[J]. Biotechnol J, 2015, 10(5): 790-800. doi: 10.1002/biot.201400764 pmid: 25740626
[16]	Lee N, Shin J, Park JH, et al. Targeted gene deletion using DNA-free RNA-guided Cas9 nuclease accelerates adaptation of CHO cells to suspension culture[J]. ACS Synth Biol, 2016, 5(11): 1211-1219. pmid: 26854539
[17]	Minkenberg B, Wheatley M, Yang YN. CRISPR/Cas9-enabled multiplex genome editing and its application[J]. Prog Mol Biol Transl Sci, 2017, 149: 111-132.
[18]	Wang Q, Betenbaugh MJ. Metabolic engineering of CHO cells to prepare glycoproteins[J]. Emerg Top Life Sci, 2018, 2(3): 433-442. doi: 10.1042/ETLS20180056 pmid: 33525787
[19]	Shin SW, Lee JS. Optimized CRISPR/Cas9 strategy for homology-directed multiple targeted integration of transgenes in CHO cells[J]. Biotechnol Bioeng, 2020, 117(6): 1895-1903. doi: 10.1002/bit.27315 pmid: 32086804
[20]	Wang WP, Zheng WY, Hu FZ, et al. Enhanced biosynthesis performance of heterologous proteins in CHO-K1 cells using CRISPR-Cas9[J]. ACS Synth Biol, 2018, 7(5): 1259-1268. doi: 10.1021/acssynbio.7b00375 pmid: 29683658
[21]	Sakuma T, Yamamoto T. Magic wands of CRISPR-lots of choices for gene knock-in[J]. Cell Biol Toxicol, 2017, 33(6): 501-505. doi: 10.1007/s10565-017-9409-6 pmid: 28828704
[22]	Shin SW, Lee JS. CHO cell line development and engineering via site-specific integration: challenges and opportunities[J]. Biotechnol Bioprocess Eng, 2020, 25(5): 633-645. doi: 10.1007/s12257-020-0093-7 URL
[23]	Yao X, Wang X, Hu XD, et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9[J]. Cell Res, 2017, 27(6): 801-814. doi: 10.1038/cr.2017.76 pmid: 28524166
[24]	Yao X, Zhang ML, Wang X, et al. Tild-CRISPR allows for efficient and precise gene knockin in mouse and human cells[J]. Dev Cell, 2018, 45(4): 526-536.e5. doi: S1534-5807(18)30327-7 pmid: 29787711
[25]	Smirnikhina SA, Anuchina AA, Lavrov AV. Ways of improving precise knock-in by genome-editing technologies[J]. Hum Genet, 2019, 138(1): 1-19. doi: 10.1007/s00439-018-1953-5 pmid: 30390160
[26]	Lee JS, Grav LM, Pedersen LE, et al. Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment[J]. Biotechnol Bioeng, 2016, 113(11): 2518-2523. doi: 10.1002/bit.26002 pmid: 27159230