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
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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.
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Hooman Kaghazian, a:1:{s:5:"en_US";s:25:"Pasteur Institute of Iran";}
Department of Research and Development, Research& Production Complex, Pasteur Institute
Seyed Mehdi Hassanzadeh
Department of Research and Development, Research& Production Complex, Pasteur Institute of Iran, Tehran
Azadeh Mirfeizollahi
Farnad Fanavar Tasnim Pharmed Company, Tehran, Iran
Jafar Nikzad
Padtan Pharmed Imen Zist Company, Tehran, Iran.