Knockout and Knockin by Precision Genome Editing

As a leading provider of glycoprotein related services, Creative Biolabs can help you establish customized cell line for glycoengineering by precision genome editing. With over a decade of experience and the state-of-the-art technical platforms, we can totally meet your project requirements and budgets in the gene knockout and knockin services.

Gene Knockout and Knockin

Gene knockout is a genetic technique that makes one or more genes inoperative in living organism. Knockout organisms are used to study the gene function, usually by investigating the deficiency effect of the gene. In post-translational modifications, studies have reported the knockin of human N-glycan-modifying enzymes to obtain non-immunogenic and/or ‘humanized’ N-glycans on glycosylated plant-made therapeutics. Gene knockout/knockin is a powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell.

Fig.1 A schematic representation of technology revolution from conventional to modern cell engineering (Dangi, 2018).

Applications in Glycoengineering

Many experimental cases demonstrate the effectiveness of gene knockout/knockin or combinational genetic manipulation in glycosylation engineering.

• Chinese Hamster Ovary (CHO) Cells

CHO cells were successfully engineered to produce rhEPO containing almost exclusively α2,6-sialylation by knockout of st3gal4/6 genes and knockin of st6gal-I gene, which resulted in considerable increase of α2,6 sialylation. Homogeneous bi-antennary N-glycans capped by α-2,6-NeuA were produced by additional knockout of mgat4A/4B/5 genes. Double knockout of fut8 and B4galt1 genes in CHO cells expressing a recombinant IgG1 resulted in homogeneous biantennary N-glycans lacking Fuc, containing minor amounts of Gal. CHO glutamine synthetase (GS)-knock out cell lines were developed. Fut8 knockout CHO cell lines have been established and one is currently available on the market for glycoprotein productions. CRISPR-Cas9 system has been used to knockout GDP-fucose transporter (GFT) gene in CHO cell line for the production of various afucosylated mAbs and Fc-fusion proteins. Other strategies include knockout of CMP-Neu5Ac hydroxylase (CMAH), α1,3-galactosyltransferase (α1,3-GalT), and so forth.

Fig.2 Summary of genetic approaches for manipulating glycosylation in CHO cells. Gene knockouts are represented in yellow and gene knock-in, over expression and alteration are represented in green (Tejwani, 2018).

• Yeast Cells

Knockout α1,6-mannosyltransferase (Och1p) in yeast has been established. Double knockout of both STE13 and DAP2 facilitating the production of recombinant glycoproteins with their intact native sequence in yeast. Transfer and elongation of O-linked mannose can be avoided by knockout of protein O-mannosyltransferases (PMTs) in yeast.

Fig.3 Glycoengineering strategies in yeasts (Fidan, 2015).

• Plant Cells

In plants, a BY2 cell line devoid of plants specific xylose and fucose by knocking out of the XylT and the FucT genes has been established. The glycoengineered BY2 cell lines provide a valuable platform for producing potent biopharmaceutical products that can be similar to the mammalian proteins. Similarly, knockout of other plant-specific N-glycan maturation enzymes, such as α1,3-fucosyltransferases (α1,3-FTs) and β1,2- xylosyltransferase (β1,2-XT), or the knockin of human N-glycan-modifying enzymes have been proved to be successful in plant cells. Unwanted plant-specific modifications also can be eliminated by knockout of the responsible enzymes like prolyl 4-hydroxylases (P4H).

• Insect Cells

In insect cells, knockout of core α1,3-fucosyltransferase (FUT) and b-N-acetylglucosaminidase (β-hex) was reported. CRISPR-Cas9 system has also been reported for editing fdl gene in S2 cells, resulting in S2 cells produced partially elongated, mammalian-type complex N-glycans.

Features of Our Knockout/Knockin Services

• Strategies for gene knockout/knockin: CRISPR/Cas9 system, transcription activator-like effector nucleases, and homologous recombination technology.
• Complete project report
• High quality and fast delivery
• Best after-sale service

Creative Biolabs offers a range of gene manipulation solutions by gene knockout and gene knockin techniques to fit your demands. Scientists in our laboratory have accumulated extensive experience in precision genome editing for site-specific gene manipulation. Please feel free to contact us for more details.

References

1. Dangi, A. K.; et al. Cell line techniques and gene editing tools for antibody production: A Review. Frontiers in pharmacology. 2018, 9, 630.
2. Tejwani, V.; et al. Glycoengineering in CHO cells: Advances in systems biology. Biotechnology journal. 2018, 13(3), 1700234.
3. Fidan, O.; Zhan, J. Recent advances in engineering yeast for pharmaceutical protein production. RSC Advances. 2015, 5(105), 86665-86674.

Related Services:

1. Knockdown
2. Overexpression
3. Manipulating Heterogeneity
4. Manipulating Sialylation
5. Manipulating Fucosylation
6. Manipulating Branching
7. Manipulating Glycosaminoglycan