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One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment.
Grav, Lise Marie; Lee, Jae Seong; Gerling, Signe; Kallehauge, Thomas Beuchert; Hansen, Anders Holmgaard; Kol, Stefan; Lee, Gyun Min; Pedersen, Lasse Ebdrup; Kildegaard, Helene Faustrup.
Afiliación
  • Grav LM; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
  • Lee JS; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
  • Gerling S; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
  • Kallehauge TB; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
  • Hansen AH; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
  • Kol S; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
  • Lee GM; Department of Biological Sciences, KAIST, Daejeon, Republic of Korea.
  • Pedersen LE; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
  • Kildegaard HF; The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. hef@biosustain.dtu.dk.
Biotechnol J ; 10(9): 1446-56, 2015 Sep.
Article en En | MEDLINE | ID: mdl-25864574
ABSTRACT
The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.
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Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Biotecnología / Proteínas Fluorescentes Verdes / Técnicas de Inactivación de Genes / Sistemas CRISPR-Cas / Citometría de Flujo Límite: Animals Idioma: En Revista: Biotechnol J Asunto de la revista: BIOTECNOLOGIA Año: 2015 Tipo del documento: Article País de afiliación: Dinamarca

Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Biotecnología / Proteínas Fluorescentes Verdes / Técnicas de Inactivación de Genes / Sistemas CRISPR-Cas / Citometría de Flujo Límite: Animals Idioma: En Revista: Biotechnol J Asunto de la revista: BIOTECNOLOGIA Año: 2015 Tipo del documento: Article País de afiliación: Dinamarca