Accelerated Homology-Directed Targeted Integration of Transgenes in Chinese Hamster Ovary Cells Via CRISPR/Cas9 and Fluorescent Enrichment

Targeted gene integration into site-specific loci can be achieved in Chinese hamster ovary (CHO) cells via CRISPR/Cas9 genome editing technology and the homology-directed repair (HDR) pathway. The low efficiency of HDR often requires antibiotic selection, which limits targeted integration of multiple genes at multiple sites. To improve HDR-mediated targeted integration, while avoiding the use of selection markers, chemical treatment for increased HDR, and fluorescent enrichment of genome-edited cells was assessed in CHO cells. Chemical treatment did not improve HDR-mediated targeted integration. In contrast, fluorescent markers in Cas9 and donor constructs enable FACS enrichment, resulting in a threefold increase in the number of cells with HDR-mediated genome editing. Combined with this enrichment method, large transgenes encoding model proteins (including an antibody) were successfully targeted integrated. This approach provides a simple and fast strategy for targeted generation of stable CHO production cell lines in a rational way. (C) 2016 Wiley Periodicals, Inc
Publisher
WILEY-BLACKWELL
Issue Date
2016-11
Language
English
Keywords

DNA-REPAIR PATHWAY; MAMMALIAN-CELLS; CHO-CELLS; RECOMBINATION; EFFICIENCY; RAD51

Citation

BIOTECHNOLOGY AND BIOENGINEERING, v.113, no.11, pp.2518 - 2523

ISSN
0006-3592
DOI
10.1002/bit.26002
URI
http://hdl.handle.net/10203/214608
Appears in Collection
BS-Journal Papers(저널논문)
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