PRECISE AND SITE-SPECIFIC EDITING OF THE CHO HOST GENOME VIA CRISPR/CAS9 FOLLOWED BY THE INTEGRASE SYSTEM TO GENERATE A RECOMBINANT ARYLSULFATASE B PRODUCING CELL LINE

Main Article Content

Hooman Kaghazian
Seyed Mehdi Hassanzadeh
Azadeh Mirfeizollahi
Jafar Nikzad

Keywords

Recombinant cell line development, arylsulfatase B enzyme, CRISPR/Cas9, GFP

Abstract

Stable recombinant cell line development by site direct integration of a gene of interest is critical issue in therapeutic proteins production such as arylsulfatase B enzyme. Deficiency of this enzyme in human body causes mucopolysaccharidosis type VI diseases which is treated by recombinant arylsulfatase B enzyme replacement. So in this work combination of homologous recombination (HR) method through clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 and integrase system were employed to precise integration of ARSB gene into the Chinese hamster ovary (CHO) host genome for stable production of recombinant enzyme. CHO-k1 cells were cultured and transfected by green fluorescent protein (GFP) donor plasmid and pX330 vector that targeted Rosa26 locus in the host genome by CRISPR/Cas9 technique. Then GFP positive cells were selected and edited through cassette exchange integrase system to convert recombinant arylsulfatase B producer cell line. Analysis of recombinant cell lines by ELISA verified arylsulfatase B enzyme expression in all GFP negative single clones after several passages. In conclusion using a precise and specific site direct integration method for genetic manipulation can lead to stability and continuous production of recombinant protein in modified cell lines.

Abstract 195 | pdf Downloads 167

References

1. Walsh G. Biopharmaceutical benchmarks 2014. Nature Biotechnology. 2014;32(10):992-1000.
2. Zhu J. Mammalian Cell Protein Expression for Biopharmaceutical Production. Biotechnol. 2012;30:1158-70.
3. D Ghaderi MZ, N Hurtado, et al. Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation. Biotechnology and Genetic Engineering Reviews 2012;28:147-76.
4. Wurm F. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 2004;22:1393-8.
5. LM Barnes CB, AJ Dickson. Stability of protein production from recombinant mammalian cells. Biotechnol Bioeng. 2003;81:631-9.
6. M Kim POC, KA Droms, et al. .A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnol Bioeng. 2011;108:2434-46.
7. S Li XG, R Peng, et al. FISH‐based analysis of clonally derived CHO cell populations reveals high probability for transgene integration in a terminal region of chromosome 1 (1q13). PLoS ONE. 2016;11.
8. Y Kawabe SK, M Murakami, M, et al. 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. Journal of Bioscience and Bioengineering. 2018;125:599-605.
9. F Zhu MG, AP Farruggio, et al DICE, an efficient system for iterative genomic editing in human pluripotent stem cells. Nucleic Acids Research. 2014;42(5).
10. L Cong ea. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819-23.
11. SW Cho SK, JM Kim. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 2013;31:230-2.
12. G Gasiunas RB, P Horvath, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA. 2012;109:2579-86.
13. M Jinek ea. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816-21.
14. "ARSB", Genetics Home Resource: U.S. National Library of Medicine; 2010.
15. J Hopwood GB, P Kirkpatrick Galsulfase. Nat Rev Drug Discov. 2006;5:101-2.
16. B P Zambrowicz AI, S Fiering, et al. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci 1997;94(8):3789-94.
17. D Biggs CC, B Davies Targeted Integration of Transgenes at the Mouse Gt(ROSA)26Sor Locus Methods Mol Biol. 2023;2631:299-323.
18. CM Chen JK, S Bhattacharya, et al. A comparison of exogenous promoter activity at the ROSA26 locus using a PhiiC31 integrase mediated cassette exchange approach in mouse ES cells. PLoS ONE. 2011;6(8).