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A Novel Bxb1 Integrase RMCE System For High Fidelity Site‐specific Integration Of MAb Expression Cassette In CHO Cells

Mara C. Inniss, Kalpanie Bandara, Barbara Jusiak, Timothy Lu, R. Weiss, Liliana Wroblewska, L. Zhang
Published 2017 · Biology, Medicine

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As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site‐specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system. We first integrated a DNA construct flanked by either Bxb1 attachment sites or FRT sequences (referred to as a landing pad) into the Fer1L4 genomic locus of CHO‐S cells using CRISPR/Cas9 mediated homologous recombination. We characterized the resulting clones harboring either the Bxb1 or Flp/FRT landing pad using whole genome resequencing to compare their genomes with the parental host cell line. We determined that each landing pad was specifically integrated into the Fer1L4 locus in the selected clones and observed no major structural changes in the genome or variations in copy number as a result of CRISPR/Cas9 modification. We subsequently tested the ability of the Bxb1 and Flp/FRT landing pad clones to perform proper RMCE with donor vectors containing identical mAb expression cassettes flanked by either Bxb1 attachment sites or FRT sites. We demonstrated that both RMCE systems were able to generate stable pools in a similar time frame with comparable mAb expression. Through genetic characterization of up to 24 clones derived from either system, we determined that the BxB1 RMCE system yielded higher fidelity RMCE events than the Flp/FRT system as evidenced by a higher percentage of clones with expected integration of the mAb cassette into the landing pad in the respective cell lines. We conclude that Bxb1 RMCE is an excellent alternative to Flp/FRT RMCE and valuable addition to our toolbox enabling the engineering of more sophisticated cell lines for biotherapeutic production. Biotechnol. Bioeng. 2017;114: 1837–1846. © 2017 Wiley Periodicals, Inc.
This paper references
10.1016/0167-4781(94)90224-0
Blasticidin S deaminase gene from Aspergillus terreus (BSD): a new drug resistance gene for transfection of mammalian cells.
M. Kimura (1994)
10.1021/BI00209A003
Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci.
T. Schlake (1994)
10.1016/S1097-2765(03)00444-1
The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB.
P. Ghosh (2003)
10.1016/S1534-5807(03)00399-X
Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice.
Catherine S. Branda (2004)
10.1021/bp049780w
On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells
S. Schlatter (2005)
10.1016/J.TIBTECH.2005.06.009
Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools.
C. Coates (2005)
10.1021/bp0501524
Regulation of Recombinant Monoclonal Antibody Production in Chinese Hamster Ovary Cells: A Comparative Study of Gene Copy Number, mRNA Level, and Protein Expression
Z. Jiang (2006)
10.1007/s00438-006-0129-5
A diversity of serine phage integrases mediate site-specific recombination in mammalian cells
A. Keravala (2006)
10.2144/000112150
Phage Bxb1 integrase mediates highly efficient site-specific recombination in mammalian cells.
J. P. Russell (2006)
10.1093/nar/gkn347
Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks
W. Mansour (2008)
10.1007/S12257-008-0151-Z
Characterization of site-specific recombination mediated by Cre recombinase during the development of erythropoietin producing CHO cell lines
M. Kim (2008)
Use of Flp-mediated cassette exchange in the development of a CHO cell line stably producing erythropoietin.
M. Kim (2008)
10.1093/bioinformatics/btp352
The Sequence Alignment/Map format and SAMtools
Heng Li (2009)
10.1093/bioinformatics/btp324
Fast and accurate short read alignment with Burrows–Wheeler transform
Heng Li (2009)
10.1007/s10616-011-9397-y
Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system
Y. Kawabe (2011)
10.1038/nbt.1754
Integrative Genomics Viewer
James T. Robinson (2011)
10.1371/journal.pone.0017267
A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector
Shigeyuki Yamaguchi (2011)
10.1038/nbt.1932
The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line
X. Xu (2011)
10.1016/j.jbiotec.2011.09.023
IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.
Steven C L Ho (2012)
10.1093/nar/gks003
cn.MOPS: mixture of Poissons for discovering copy number variations in next-generation sequencing data with a low false discovery rate
G. Klambauer (2012)
10.1002/btpr.1783
Fast identification of reliable hosts for targeted cell line development from a limited‐genome screening using combined φC31 integrase and CRE‐Lox technologies
Y. Crawford (2013)
10.1016/j.gene.2012.11.016
Recombinase-mediated cassette exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications.
Soeren Turan (2013)
10.1186/1472-6750-13-87
Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome
Zhengyao Xu (2013)
10.1126/science.1232033
RNA-Guided Human Genome Engineering via Cas9
P. Mali (2013)
10.1093/bib/bbs017
Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration
H. Thorvaldsdóttir (2013)
10.1002/0471250953.bi1112s47
BEDTools: The Swiss‐Army Tool for Genome Feature Analysis
Aaron R. Quinlan (2014)
10.1093/bioinformatics/btu393
circlize implements and enhances circular visualization in R
Z. Gu (2014)
10.1093/nar/gkt1290
DICE, an efficient system for iterative genomic editing in human pluripotent stem cells
F. Zhu (2014)
10.1038/ncomms6560
Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9
Shota Nakade (2014)
10.1101/005074
CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes
John Aach (2014)
10.1093/nar/gku1082
A platform for rapid prototyping of synthetic gene networks in mammalian cells
X. Duportet (2014)
10.1038/nbt.3155
Inducible in vivo genome editing with CRISPR/Cas9
L. Dow (2015)
10.1002/btpr.2175
Recombinase‐mediated cassette exchange (RMCE) for monoclonal antibody expression in the commercially relevant CHOK1SV cell line
L. Zhang (2015)
10.1038/nchembio.1736
Modular construction of mammalian gene circuits using TALE transcriptional repressors
Yinqing Li (2015)
10.1016/j.stemcr.2015.04.016
A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells
Zengrong Zhu (2015)
10.1002/biot.201500027
One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment.
L. M. Grav (2015)
10.3390/ijms161023849
Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids
T. Sakuma (2015)
10.1038/srep08572
Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway
J. S. Lee (2015)
10.1186/s12896-016-0241-5
Comparison and optimization of ten phage encoded serine integrases for genome engineering in Saccharomyces cerevisiae
Zhengyao Xu (2016)
10.1002/bit.26002
Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment.
J. S. Lee (2016)



This paper is referenced by
10.1016/j.jbiotec.2021.06.018
Targeted Integration into Pseudo attP Sites of CHO Cells Using CRISPR/Cas9.
Sana Pourtabatabaei (2021)
10.1002/btpr.3158
Genetic rearrangement during site specific integration event facilitates cell line development of a bispecific molecule
Barbara Tevelev (2021)
10.1016/J.DDTEC.2021.02.003
Recent advances in CHO cell line development for recombinant protein production
B. Tihanyi (2021)
10.1002/9783527823468.CH10
Genome Editing of Eukarya
Jonathan A. Arnesen (2021)
10.1051/MATECCONF/202133307002
Retrotransposon-mediated Gene Transfer for Animal Cells
Feiyan Zheng (2021)
10.1002/btpr.3140
Development of a targeted integration Chinese hamster ovary host directly targeting either one or two vectors simultaneously to a single locus using the Cre/Lox recombinase‐mediated cassette exchange system
Domingos Ng (2021)
10.1002/bit.27701
Epigenomic features revealed by ATAC‐seq impact transgene expression in CHO cells
Zion Lee (2021)
10.1002/btpr.3137
CHOK1SV GS‐KO SSI expression system: A combination of the Fer1L4 locus and glutamine synthetase selection
M. Feary (2021)
10.1051/MATECCONF/202133307001
Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems
Ryusei Iwao (2021)
10.1021/acssynbio.0c00322
Multi-copy targeted integration for accelerated development of high-producing CHO cells.
D. Sergeeva (2020)
10.1080/07388551.2020.1768043
The use of site-specific recombination and cassette exchange technologies for monoclonal antibody production in Chinese Hamster ovary cells: retrospective analysis and future directions
K. Srirangan (2020)
10.1007/s12257-020-0093-7
CHO Cell Line Development and Engineering via Site-specific Integration: Challenges and Opportunities
S. Shin (2020)
10.1002/biot.202000057
A Site-Specific Integration Reporter System that Enables Rapid Evaluation of CRISPR/Cas9-mediated Genome Editing Strategies in CHO Cells.
Nathaniel K Hamaker (2020)
10.1002/BIT.27315
Optimized CRISPR/Cas9 strategy for homology‐directed multiple targeted integration of transgenes in CHO cells
S. Shin (2020)
10.1002/btpr.2967
Maximizing Antibody Production in a Targeted Integration Host by Optimization of Subunit Gene Dosage and Position.
Joe Carver (2020)
10.1016/j.coche.2020.06.003
Synthetic biology approaches: the next tools for improved protein production from CHO cells
Claire E McGraw (2020)
10.1002/bit.27599
Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis
William Hilliard (2020)
10.1101/2020.12.11.420000
A Synthetic Transcription Platform for Programmable Gene Expression in Mammalian Cells
William C W Chen (2020)
10.1016/j.coche.2020.100663
Cell culture bioprocessing - the road taken and the path forward.
S. O’Brien (2020)
10.1002/bit.26823
Recurring genomic structural variation leads to clonal instability and loss of productivity
Arpan A Bandyopadhyay (2019)
10.1007/s12247-019-09411-6
Application of Genetic Engineering in Biotherapeutics Development
Patrick G Schweickert (2019)
10.1002/9783527811410.ch8
CRISPR Toolbox for Mammalian Cell Engineering
D. Sergeeva (2019)
10.1002/btpr.2772
Utilizing a regulated targeted integration cell line development approach to systematically investigate what makes an antibody difficult to express
Tomofumi Tadauchi (2019)
10.1007/s00253-018-9021-6
Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35
Menglin Zhao (2018)
10.1016/J.COCHE.2018.09.010
Beyond preclinical research: production of CHO-derived biotherapeutics for toxicology and early-phase trials by transient gene expression or stable pools
Matthew Stuible (2018)
10.1093/nar/gky216
A multi-landing pad DNA integration platform for mammalian cell engineering
L. Gaidukov (2018)
10.1021/acs.analchem.7b05007
Proteins and Proteoforms: New Separation Challenges.
F. Regnier (2018)
10.1007/s10295-018-2027-3
Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications
Mitchell S Weisenberger (2018)
10.1016/j.tibtech.2018.04.006
Exploiting Bacteriophage Proteomes: The Hidden Biotechnological Potential.
S. Santos (2018)
10.1016/j.jbiosc.2017.12.003
Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems.
Y. Kawabe (2018)
Developing Tools for Improved and Predictable Recombinant Protein Production in CHO Cells
Nuša Pristovšek (2018)
10.1186/s12919-018-0097-x
Abstracts from the 25th European Society for Animal Cell Technology Meeting: Cell Technologies for Innovative Therapies
Holger Laux (2018)
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