Nexcelom’s Celigo Detects CRISPR Transfection Efficiency of sgRNA in CHO Cells

Introduction

Chinese hamster ovary (CHO) cells are the cell type of choice for biopharmaceuticals due to their propensity to correctly fold and post-translationally modify human proteins [1]. Here, researchers use an optimized bioinformatics tool (“CRISPy”) to generate chimeric RNA called “single guide” RNA (sgRNA) in order to disrupt FUT8, a gene that is utilized in N-glycosylation. This novel web-based tool ensures efficient, fast, and low-cost genetic manipulation of CHO cells for future biopharmaceutical applications.

Materials and Methods

Cell culture and transfection of CHO cells

CHO cells were cultured and transfected by Nucleofector Kit V with 1 μg of Cas9 plasmid and 1 μg of sgRNA plasmid. Two days later, cells were transfected with pmaxGFP and assessed for transfection efficiency using Nexcelom’s Celigo.

Phenotypic analysis of FUT8 knockout cells

Five days post-transfection, the selection agent Lens culinaris agglutinin (LCA) was added. (LCA binds plasma membrane proteins, which leads to downstream cell death. LCA serves as a selection agent for FUT8 knockout cells as LCA cannot bind to cells devoid of FUT8.) Cells were stained for phenotypic analysis with Hoechst (red in image) and fluorescein-labeled LCA (F-LCA; green in image). FUT8 wild-type cells will stain green, while FUT8 knockout cells will not. Bright-field and fluorescent images were captured with the Celigo.

*Note:  For more detailed Materials and Methods and a complete account of the entire study, please refer to the original manuscript [http://onlinelibrary.wiley.com/doi/10.1002/bit.25233/abstract]

Results

  • LCA selection caused control cells to become non-adherent and rounded.
  • Cells transfected with Cas9 and sgRNA remained adherent, indicating the successful knockout of FUT8 in those cells.
  • Cells transfected with Cas9 and sgRNA without LCA selection showed that F-LCA negative cells represented 29.1% of the entire population.
  • Cells transfected with Cas9 and sgRNA with LCA selection showed 98.6% of cells were F-LCA negative, as demonstrated by other studies proving the lack of functional FUT8 [2-4].
Figure 1A-C. Phenotypic analysis of FUT8 knockout cells. Cells were transfected with either Cas9 plasmid alone or Cas9 plasmid plus sgRNA. At Day 6 the bright field images were taken (A). On Day 13 the cells were stained with Hoechst (red) and fluorescein-labeled LCA (green). Quantification of the images for F-LCA positive (wild-type FUT8) and F-LCA negative (FUT8 knockout) can be seen in C.

Figure 1A-C. Phenotypic analysis of FUT8 knockout cells. Cells were transfected with either Cas9 plasmid alone or Cas9 plasmid plus sgRNA. At Day 6 the bright field images were taken (A). On Day 13 the cells were stained with Hoechst (red) and fluorescein-labeled LCA (green). Quantification of the images for F-LCA positive (wild-type FUT8) and F-LCA negative (FUT8 knockout) can be seen in C.

Conclusions

  • This RNA-guided CRISPR Cas9 application successfully disrupted genes in CHO cells.
  • The web-based bioinformatics design tool “CRISPy” provides an effective, low-cost method for genetically manipulating CHO cells.

References

  1. Jayapal, K.P., et al., Recombinant protein therapeutics from CHO cells – 20 years and counting. Chem Eng Prog, 2007. 103: p. 40-47.
  2. Malphettes, L., et al., Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies. Biotechnol Bioeng, 2010. 106(5): p. 774-83.
  3. Mori, K., et al., Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA. Biotechnol Bioeng, 2004. 88(7): p. 901-8.
  4. Yamane-Ohnuki, N., et al., Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng, 2004. 87(5): p. 614-22.

 

By |2015-03-30T09:00:16+00:00March 30th, 2015|Categories: Celigo User Publications|Tags: , , , , |0 Comments

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