Recombinase-mediated Site-specific Integration and Its Application in the Construction of Recombinant CHO Cells

doi:10.13523/j.cb.2207027

China Biotechnology

2022, Vol. 42

Issue (12): 52-60 DOI: 10.13523/j.cb.2207027

Recombinase-mediated Site-specific Integration and Its Application in the Construction of Recombinant CHO Cells

SUN Jin-yu^1,²,LIU Guang²,LI Chen³,WANG Ying^1,^*(

),LIU Guo-qing^2,^*(

)

1 College of Life Science and Technology, China Pharmaceutical University, Nanjing 211198, China
2 Shanghai Henlius Biotech, Inc., Shanghai 200233, China
3 Aton (Shanghai) Biotech Co., Ltd., Shanghai 201620, China

Download:

HTML

PDF(631KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Chinese Hamster Ovary (CHO) cells are the most commonly used cells for producing therapeutic recombinant proteins. At present, random integration (RI) is still the main strategy for the construction of recombinant CHO cell lines. Due to the lack of genomic stability in CHO cells, 1-2 rounds of high-throughput screening are usually required to obtain cell lines with high yield, good quality and suitable for a specific process,which is a process with heavy workload, time-consuming and poor batch stability. Site-specific integration (SSI) is a gene editing technique that integrates foreign genes into specific sites to obtain stable, high-yield cell lines adapted to specific production process through one round of screening, thus shortening the construction cycle of cell lines. In recent years, there have been continuous reports of cases where the site-specific integration strategy was applied to construct recombinant CHO cell lines. The most commonly used techniques are nuclease, transposon, and recombinase techniques. The recombinase with great commercial prospect and its application in CHO cell line construction are discussed after considering the construction process, efficiency and patents.

Key words： Gene editing Site-specific integration Recombinase CHO Recombination-mediated cassette exchange (RMCE)

Received: 14 July 2022 Published: 05 January 2023

ZTFLH:

Q819

Corresponding Authors: Ying WANG,Guo-qing LIU E-mail: waying@CPU.edu.cn;Guoqing_Liu@henlius.com

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Jin-yu SUN
	Guang LIU
	Chen LI
	Ying WANG
	Guo-qing LIU

Cite this article:

SUN Jin-yu,LIU Guang,LI Chen,WANG Ying,LIU Guo-qing. Recombinase-mediated Site-specific Integration and Its Application in the Construction of Recombinant CHO Cells. China Biotechnology, 2022, 42(12): 52-60.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2207027 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I12/52

Fig.1 Recombinase-mediated integration, excision and inversion

Table 1 Comparison of three gene editing technologies

Fig.2 RMCE reaction mediated by tyrosine recombinase (a) and serine recombinase (b)


[1]	Tihanyi B, Nyitray L. Recent advances in CHO cell line development for recombinant protein production. Drug Discovery Today: Technologies, 2020, 38: 25-34. doi: 10.1016/j.ddtec.2021.02.003 pmid: 34895638

[2]	Zhang H Y, Fan Z L, Wang T Y. Advances of glycometabolism engineering in Chinese hamster ovary cells. Frontiers in Bioengineering and Biotechnology, 2021, 9: 774175. doi: 10.3389/fbioe.2021.774175

[3]	Lalonde M E, Durocher Y. Therapeutic glycoprotein production in mammalian cells. Journal of Biotechnology, 2017, 251: 128-140. doi: 10.1016/j.jbiotec.2017.04.028

[4]	Donini R, Haslam S M, Kontoravdi C. Glycoengineering Chinese hamster ovary cells: a short history. Biochemical Society Transactions, 2021, 49(2): 915-931. doi: 10.1042/BST20200840 pmid: 33704400

[5]	Mahé A, Martiné A, Fagète S, et al. Exploring the limits of conventional small-scale CHO fed-batch for accelerated on demand monoclonal antibody production. Bioprocess and Biosystems Engineering, 2022, 45(2): 297-307. doi: 10.1007/s00449-021-02657-w

[6]	Lee J S, Kallehauge T B, Pedersen L E, et al. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Scientific Reports, 2015, 5: 8572. doi: 10.1038/srep08572 pmid: 25712033

[7]	Baumann M, Gludovacz E, Sealover N, et al. Preselection of recombinant gene integration sites enabling high transcription rates in CHO cells using alternate start codons and recombinase mediated cassette exchange. Biotechnology and Bioengineering, 2017, 114(11): 2616-2627. doi: 10.1002/bit.26388 pmid: 28734047

[8]	Thomas K R, Folger K R, Capecchi M R. High frequency targeting of genes to specific sites in the mammalian genome. Cell, 1986, 44(3): 419-428. pmid: 3002636

[9]	Jasin M, Berg P. Homologous integration in mammalian cells without target gene selection. Genes & Development, 1988, 2(11): 1353-1363. doi: 10.1101/gad.2.11.1353

[10]	Rouet P, Smih F, Jasin M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. PNAS, 1994, 91(13): 6064-6068. doi: 10.1073/pnas.91.13.6064 pmid: 8016116

[11]	Capecchi M R. The origin and evolution of gene targeting. Developmental Biology, 2022, 481: 179-187. doi: 10.1016/j.ydbio.2021.10.007

[12]	McClintock B. The origin and behavior of mutable loci in maize. PNAS, 1950, 36(6): 344-355. doi: 10.1073/pnas.36.6.344 pmid: 15430309

[13]	Ivics Z, Hackett P B, Plasterk R H, et al. Molecular reconstruction of sleeping beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell, 1997, 91(4): 501-510. doi: 10.1016/s0092-8674(00)80436-5 pmid: 9390559

[14]	Akopian A, Stark W M. Site-specific DNA recombinases as instruments for genomic surgery. Advances in Genetics, 2005, 55: 1-23. pmid: 16291210

[15]	Nunes-Düby S E, Kwon H J, Tirumalai R S, et al. Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Research, 1998, 26(2): 391-406. pmid: 9421491

[16]	Salava H, Thula S, Mohan V, et al. Application of genome editing in tomato breeding: mechanisms, advances, and prospects. International Journal of Molecular Sciences, 2021, 22(2): 682. doi: 10.3390/ijms22020682

[17]	Zhang B H. CRISPR/Cas9: a robust genome-editing tool with versatile functions and endless application. International Journal of Molecular Sciences, 2020, 21(14): 5111. doi: 10.3390/ijms21145111

[18]	Kim S, Kim J S. Targeted genome engineering via zinc finger nucleases. Plant Biotechnology Reports, 2011, 5(1): 9-17. pmid: 21837253

[19]	Joung J K, Sander J D. TALENs: a widely applicable technology for targeted genome editing. Nature Reviews Molecular Cell Biology, 2013, 14(1): 49-55. doi: 10.1038/nrm3486 pmid: 23169466

[20]	Richter C, Chang J T, Fineran P C. Function and regulation of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. Viruses, 2012, 4(10): 2291-2311. doi: 10.3390/v4102291

[21]	Beerli R R, Barbas C F. Engineering polydactyl zinc-finger transcription factors. Nature Biotechnology, 2002, 20(2): 135-141. pmid: 11821858

[22]	Janik E, Niemcewicz M, Ceremuga M, et al. Various aspects of a gene editing system-CRISPR-Cas9. International Journal of Molecular Sciences, 2020, 21(24): 9604. doi: 10.3390/ijms21249604

[23]	Zhang F, Wen Y, Guo X. CRISPR/Cas9 for genome editing: progress, implications and challenges. Human Molecular Genetics, 2014, 23(R1): R40-R46. doi: 10.1093/hmg/ddu125

[24]	Banan M. Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells. Journal of Biotechnology, 2020, 308: 1-9. doi: S0168-1656(19)30920-4 pmid: 31751596

[25]	Lentsch E, Li L, Pfeffer S, et al. CRISPR/Cas9-mediated knock-out of KrasG12D mutated pancreatic cancer cell lines. International Journal of Molecular Sciences, 2019, 20(22): 5706. doi: 10.3390/ijms20225706

[26]	Babakhani S, Oloomi M. Transposons: the agents of antibiotic resistance in bacteria. Journal of Basic Microbiology, 2018, 58(11): 905-917. doi: 10.1002/jobm.201800204 pmid: 30113080

[27]	Sandoval-Villegas N, Nurieva W, Amberger M, et al. Contemporary transposon tools: a review and guide through mechanisms and applications of Sleeping Beauty, piggyBac and Tol2 for genome engineering. International Journal of Molecular Sciences, 2021, 22(10): 5084. doi: 10.3390/ijms22105084

[28]	Bodea G O, McKelvey E G Z, Faulkner G J. Retrotransposon-induced mosaicism in the neural genome. Open Biology, 2018, 8(7): 180074. doi: 10.1098/rsob.180074

[29]	Schertzer M D, Thulson E, Braceros K C A, et al. A piggyBac-based toolkit for inducible genome editing in mammalian cells. RNA, 2019, 25(8): 1047-1058. doi: 10.1261/rna.068932.118 pmid: 31101683

[30]	Golic K G, Lindquist S. The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell, 1989, 59(3): 499-509. pmid: 2509077

[31]	Araki H, Nakanishi N, Evans B R, et al. Site-specific recombinase, R, encoded by yeast plasmid pSR1. Journal of Molecular Biology, 1992, 225(1): 25-37. pmid: 1583692

[32]	Sauer B. Inducible gene targeting in mice using the Cre/loxSystem. Methods, 1998, 14(4): 381-392. pmid: 9608509

[33]	Sauer B, McDermott J. DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages. Nucleic Acids Research, 2004, 32(20): 6086-6095. doi: 10.1093/nar/gkh941 pmid: 15550568

[34]	Ingham C J, Crombie H J, Bruton C J, et al. Multiple novel promoters from the early region in the Streptomyces temperate phage φC 31 are activated during lytic development. Molecular Microbiology, 1993, 9(6): 1267-1274. pmid: 7934940

[35]	Bibb L A, Hatfull G F. Integration and excision of the Mycobacterium tuberculosis prophage-like element, φRv1. Molecular Microbiology, 2002, 45(6): 1515-1526. doi: 10.1046/j.1365-2958.2002.03130.x

[36]	Christiansen B, Johnsen M G, Stenby E, et al. Characterization of the lactococcal temperate phage TP901-1 and its site-specific integration. Journal of Bacteriology, 1994, 176(4): 1069-1076. pmid: 8106318

[37]	Kim A I, Ghosh P, Aaron M A, et al. Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene. Molecular Microbiology, 2003, 50(2): 463-473. doi: 10.1046/j.1365-2958.2003.03723.x

[38]	Smith M C, Brown W R, McEwan A R, et al. Site-specific recombination by phiC 31 integrase and other large serine recombinases. Biochemical Society Transactions, 2010, 38(2): 388-394. doi: 10.1042/BST0380388

[39]	Meinke G, Bohm A, Hauber J, et al. Cre recombinase and other tyrosine recombinases. Chemical Reviews, 2016, 116(20): 12785-12820. doi: 10.1021/acs.chemrev.6b00077 pmid: 27163859

[40]	Feng S Q, Lu S, Grueber W B, et al. Scarless engineering of the Drosophila genome near any site-specific integration site. Genetics, 2021, 217(3): iyab012. doi: 10.1093/genetics/iyab012

[41]	Chaikind B, Bessen J L, Thompson D B, et al. A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucleic Acids Research, 2016, 44(20): 9758-9770. pmid: 27515511

[42]	O’Gorman S, Fox D T, Wahl G M. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science, 1991, 251(4999): 1351-1355. pmid: 1900642

[43]	Fukushige S, Sauer B. Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. PNAS, 1992, 89(17): 7905-7909. pmid: 1518811

[44]	Srirangan K, Loignon M, Durocher Y. The use of site-specific recombination and cassette exchange technologies for monoclonal antibody production in Chinese hamster ovary cells: retrospective analysis and future directions. Critical Reviews in Biotechnology, 2020, 40(6): 833-851. doi: 10.1080/07388551.2020.1768043 pmid: 32456474

[45]	Thyagarajan B, Olivares E C, Hollis R P, et al. Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase. Molecular and Cellular Biology, 2001, 21(12): 3926-3934. doi: 10.1128/MCB.21.12.3926-3934.2001 pmid: 11359900

[46]	Sekhavati M H, Hosseini S M, Tahmoorespur M, et al. PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection. Cell Journal, 2018, 20(1): 98-107. doi: 10.22074/cellj.2018.4385 pmid: 29308625

[47]	Naddafi F, Mahboudi F, Tabarzad M, et al. The epigenetic regulation of blinatumomab gene expression: tumor cell-dependent T cell response against lymphoma cells and cytotoxic activity. International Journal of Molecular and Cellular Medicine, 2019, 8(1): 55-66. doi: 10.22088/IJMCM.BUMS.8.1.55 pmid: 32195205

[48]	Luo J, Shen P P, Chen J. A modular toolset of phiC31-based fluorescent protein tagging vectors for Drosophila. Fly, 2019, 13(1-4): 29-41. doi: 10.1080/19336934.2019.1595999

[49]	Bernabé-Orts J M, Quijano-Rubio A, Vazquez-Vilar M, et al. A memory switch for plant synthetic biology based on the phage φC 31 integration system. Nucleic Acids Research, 2020, 48(6): 3379-3394. doi: 10.1093/nar/gkaa104 pmid: 32083668

[50]	Wang F, Ji Y T, Tian C, et al. An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase. Insect Science, 2021, 28(5): 1277-1289. doi: 10.1111/1744-7917.12866

[51]	Ata-Abadi N S, Forouzanfar M, Dormiani K, et al. Site-specific integration as an efficient method for production of recombinant human hyaluronidase PH 20 in semi-adherent cells. Applied Microbiology and Biotechnology, 2022, 106(4): 1459-1473. doi: 10.1007/s00253-022-11794-5 pmid: 35107633

[52]	Turan S, Zehe C, Kuehle J, et al. Recombinase-mediated cassette exchange (RMCE)-a rapidly-expanding toolbox for targeted genomic modifications. Gene, 2013, 515(1): 1-27. doi: 10.1016/j.gene.2012.11.016

[53]	Inniss M C, Bandara K, Jusiak B, et al. A novel Bxb 1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassette in CHO Cells. Biotechnology and Bioengineering, 2017, 114(8): 1837-1846. doi: 10.1002/bit.26268 pmid: 28186334

[54]	Scarcelli J J, Shang T Q, Iskra T, et al. Strategic deployment of CHO expression platforms to deliver Pfizer’s monoclonal antibody portfolio. Biotechnology Progress, 2017, 33(6): 1463-1467. doi: 10.1002/btpr.2493 pmid: 28480558

[55]	Sato M, Nakamura S, Inada E, et al. Recent advances in the production of genome-edited rats. International Journal of Molecular Sciences, 2022, 23(5): 2548. doi: 10.3390/ijms23052548

[56]	Chi X L, Zheng Q, Jiang R H, et al. A system for site-specific integration of transgenes in mammalian cells. PLoS One, 2019, 14(7): e0219842. doi: 10.1371/journal.pone.0219842

[57]	Li J X, Li Y J, Pawlik K M, et al. A CRISPR-Cas9, Cre-lox, and Flp-FRT cascade strategy for the precise and efficient integration of exogenous DNA into cellular genomes. The CRISPR Journal, 2020, 3(6): 470-486. doi: 10.1089/crispr.2020.0042

[58]	Dafhnis-Calas F, Xu Z Y, Haines S, et al. Iterative in vivo assembly of large and complex transgenes by combining the activities of φC 31 integrase and Cre recombinase. Nucleic Acids Research, 2005, 33(22): e189. doi: 10.1093/nar/gni192 pmid: 16361264

[59]	Lee N C O, Kim J H, Petrov N S, et al. Method to assemble genomic DNA fragments or genes on human artificial chromosome with regulated kinetochore using a multi-integrase system. ACS Synthetic Biology, 2018, 7(1): 63-74. doi: 10.1021/acssynbio.7b00209 pmid: 28799737

[60]	Moralli D, Monaco Z L. Gene expressing human artificial chromosome vectors: advantages and challenges for gene therapy. Experimental Cell Research, 2020, 390(1): 111931. doi: 10.1016/j.yexcr.2020.111931

[61]	Ahmadi M, Mahboudi F, Akbari Eidgahi M R, et al. Evaluating the efficiency of phiC 31 integrase-mediated monoclonal antibody expression in CHO cells. Biotechnology Progress, 2016, 32(6): 1570-1576. doi: 10.1002/btpr.2362

[62]	Nehlsen K, Schucht R, da Gama-Norton L, et al. Recombinant protein expression by targeting pre-selected chromosomal loci. BMC Biotechnology, 2009, 9: 100. doi: 10.1186/1472-6750-9-100 pmid: 20003421

[63]	Zhang L, Inniss M C, Han S, et al. Recombinase-mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line. Biotechnology Progress, 2015, 31(6): 1645-1656. doi: 10.1002/btpr.2175 pmid: 26399954

[64]	Feary M, Moffat M A, Casperson G F, et al. CHOK1SV GS-KO SSI expression system: a combination of the Fer1L4 locus and glutamine synthetase selection. Biotechnology Progress, 2021, 37(4): e3137. doi: 10.1002/btpr.3137 pmid: 33609084

[65]	Jusiak B, Jagtap K, Gaidukov L, et al. Comparison of integrases identifies Bxb1-GA mutant as the most efficient site-specific integrase system in mammalian cells. ACS Synthetic Biology, 2019, 8(1): 16-24. doi: 10.1021/acssynbio.8b00089 pmid: 30609349

[66]	Irion S, Luche H, Gadue P, et al. Identification and targeting of the ROSA 26 locus in human embryonic stem cells. Nature Biotechnology, 2007, 25(12): 1477-1482. doi: 10.1038/nbt1362

[67]	Gaidukov L, Wroblewska L, Teague B, et al. A multi-landing pad DNA integration platform for mammalian cell engineering. Nucleic Acids Research, 2018, 46(8): 4072-4086. doi: 10.1093/nar/gky216 pmid: 29617873

[1]	DENG Xiang-zi, ZHOU Wen-xu, LU Yan-du. *Production of 24- Methylenecholesterol from Royal Jelly by Nannocloropsis oceanica* IMET1**[J]. China Biotechnology, 2022, 42(8): 30-39.

[2]	REN Zi-qiang,WANG Meng-can,ZHANG Hai-ling,ZHU Xi-qiang,LIN Jian. Research Progress of Glycoprotein Expression in CHO Cells[J]. China Biotechnology, 2022, 42(6): 54-65.

[3]	QU Li-li,DING Xue-feng,CAI Yan-fei,LU Chen,LI Hua-zhong,JIN Jian,CHEN Yun. Discovery of Stable Expression Sites in CHO Genome NW-003614092.1[J]. China Biotechnology, 2022, 42(6): 12-19.

[4]	LIU Ming-zhu,ZHANG Liang,GUO Fang,LI Chun,FENG Xu-dong. Construction of Yeast Surface Display System and Application in Cellulose Degrading[J]. China Biotechnology, 2022, 42(5): 91-99.

[5]	ZHANG Jun-you,WANG Qi-lin,LIU Qian,QI Si-han,LI Chun-yan. Research Progress of CRISPR/Cas Gene Editing Technology in Enhancer Function Analysis and Identification[J]. China Biotechnology, 2022, 42(4): 24-32.

[6]	LIN Jian-fen, LUO Shun. Research Progress on Stable Hot Spot Sites in the Genome of CHO Cells[J]. China Biotechnology, 2022, 42(3): 72-81.

[7]	ZHANG Yi, WANG Chen, SHI Jing-jing, CHEN Xue-jun, ZHANG Rui-hua, JIN Qian, SHI Tong, LI Li-qin. Establishment of Human GABA_AR-CHO Cell Line Stable Expression[J]. China Biotechnology, 2022, 42(3): 38-46.

[8]	Xiang-zhi LIU,Chi CHENG,Yue ZHAO,Chao-jun WANG,Ying ZHANG,Chuang XUE. Induction and Regulation of Cellulase Synthesis in Trichoderma reesei[J]. China Biotechnology, 2022, 42(10): 93-104.

[9]	ZHAO Yuan-heng,GUO Jia,CHEN Liu-biao,WANG Jun-jie. A Review on Bio-preservation at Sub-freezing Temperatures under Isochoric Conditions[J]. China Biotechnology, 2022, 42(1/2): 119-127.

[10]	GUO Man-man,TIAN Kai-ren,QIAO Jian-jun,LI Yan-ni. Application of Phage Recombinase Systems in Synthetic Biology[J]. China Biotechnology, 2021, 41(8): 90-102.

[11]	MA Qiao-ni,WANG Meng,ZHU Xing-quan. Research Advances in Recombinase-aided Amplification Technology and Its Application in Rapid Detection of Pathogenic Microorganisms[J]. China Biotechnology, 2021, 41(6): 45-49.

[12]	HU Xuan,WANG Song,YU Xue-ling,ZHANG Xiao-peng. Construction of a Destabilized EGFP Cell Model for Gene Editing Evaluation[J]. China Biotechnology, 2021, 41(5): 17-26.

[13]	DUAN Yang-yang,ZHANG Feng-ting,CHENG Jiang,SHI Jin,YANG Juan,LI Hai-ning. The Effect of SIRT2 on Apoptosis and Mitochondrial Function in Parkinson’s Disease Model Cells Induced by MPP⁺[J]. China Biotechnology, 2021, 41(4): 1-8.

[14]	CAI Run-ze,WANG Zheng-bo,CHEN Yong-chang. *Research Progress of Mecp2* Affecting Metabolic Function in Rett Syndrome**[J]. China Biotechnology, 2021, 41(2/3): 89-97.

[15]	LENG Yan,SUN Kang-tai,LIU Qian-qian,PU A-qing,LI Xiang,WAN Xiang-yuan,WEI Xun. Trends of Global Gene-edited Crops Supervision[J]. China Biotechnology, 2021, 41(12): 24-29.

Viewed

Full text

Abstract

Cited

Shared

Discussed