title: Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment.
abstract: 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.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, 2970, Denmark.
authors.forename: Jae Seong
authors.initials: JS
authors.lastname: Lee
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, 2970, Denmark.
authors.forename: Lise Marie
authors.initials: LM
authors.lastname: Grav
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, 2970, Denmark.
authors.forename: Lasse Ebdrup
authors.initials: LE
authors.lastname: Pedersen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, 2970, Denmark.|Department of Biological Sciences, KAIST, Yuseong-gu, Daejeon, Republic of Korea.
authors.forename: Gyun Min
authors.initials: GM
authors.lastname: Lee
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, 2970, Denmark. hef@biosustain.dtu.dk.
authors.forename: Helene Faustrup
authors.initials: HF
authors.lastname: Kildegaard
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques / Q000379:methods; D016466:CHO Cells / Q000502:physiology*; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D018390:Gene Targeting / Q000379:methods; D024861:Genetic Enhancement / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D019076:Transgenes / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D005456:Fluorescent Dyes
keywords: CRISPR/Cas9; Chinese hamster ovary cells; fluorescent enrichment; homology-directed repair; targeted integration