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Accelerated homology‐directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment
Author(s) -
Lee Jae Seong,
Grav Lise Marie,
Pedersen Lasse Ebdrup,
Lee Gyun Min,
Kildegaard Helene Faustrup
Publication year - 2016
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26002
Subject(s) - chinese hamster ovary cell , crispr , transgene , biology , green fluorescent protein , cas9 , homology (biology) , chinese hamster , fluorescence , genetics , microbiology and biotechnology , computational biology , gene , dna , cell culture , physics , quantum mechanics
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. Biotechnol. Bioeng. 2016;113: 2518–2523. © 2016 Wiley Periodicals, Inc.