Abstract
Objective
To inspect the feasibility of recombinant stable HEK293 cell lines development for biopharmaceuticals production using CRISPR/Cas9-mediated site-specific integration.
Results
Using EGFP as a model protein, we first confirmed that the ‘safe harbor’ AAVS1 locus could be successfully targeted and the exogenous genes could be integrated through homology-directed repair induced by CRISPR/Cas9 technology. Then we constructed a donor plasmid harboring CTLA4Ig gene with an upstream CMV promoter and a downstream puromycin N-acetyltransferase gene to accelerate the efficient integration and selection of CTLA4Ig expression clones. After puromycin enrichment, the transfected pool was diluted for single clone selection, and 12 recombinant clones with CTLA4Ig expression were finally selected with a targeting efficiency of 25.8%. Productivity assay demonstrated that a frequency of 83.3% of selected clone were of consistent productivities, thus illustrating the high efficiency and success rate of this strategy.
Conclusions
CRISPR/Cas9 mediated site-specific integration is an efficient and reliable tool to establishment recombinant stable HEK293 cell lines for both academic and industrial applications.
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Acknowledgements
This work was supported in part by the Science & Technology Commission of Shanghai Municipality (Grant Nos. 17ZR1413700 and 17431904500) and Medical and Engineering Cross Research Foundation of Shanghai Jiao Tong University (Grant No. YG2016QN27).
Supporting information
Supplementary Table S1—PCR primers sequences.
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Yang, H., Wang, J., Zhao, M. et al. Feasible development of stable HEK293 clones by CRISPR/Cas9-mediated site-specific integration for biopharmaceuticals production. Biotechnol Lett 41, 941–950 (2019). https://doi.org/10.1007/s10529-019-02702-5
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DOI: https://doi.org/10.1007/s10529-019-02702-5