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Hooman Kaghazian
Seyed Mehdi Hassanzadeh
Azadeh Mirfeizollahi
Jafar Nikzad


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


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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