CRISPR-Based Strategies to Boost Production Yield in CHO Cell Lines    

Biopharmaceuticals are products extracted from biological sources and manufactured using biotechnological methods, including antibodies, cytokines, hormones, growth factors, vaccines, and peptides. Chinese Hamster Ovary (CHO) cells are the preferred mammalian cell line in biopharmaceutical production.

Advantages of CHO Cells

CHO cells are favored for several reasons. They can produce human-like proteins with complex post-translational modifications (PTMs), ensuring the biological functionality of therapeutic proteins. They can achieve gram-per-liter levels of recombinant protein production and demonstrate robust resistance to variations in oxygen levels, temperature, pressure, or pH during production. CHO cells can also be cultured in suspension and chemically defined, serum-free media, facilitating large-scale and reproducible manufacturing. Their resistance to human viruses enhances biosafety during production.

Genetic Engineering of CHO Cells

Owing to these benefits, the biopharmaceutical industry has been using CHO cells extensively for decades, building a thorough genetic toolkit and regulatory knowledge in the process. This context has prompted attempts to enhance the synthesis of biopharmaceuticals by genetically modifying CHO cells. The CRISPR-Cas system represents a major breakthrough in this sector. CRISPR technology has a number of benefits over conventional genome editing instruments like Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), including cost-effectiveness, programmability, ease of use, and increased efficiency.

CRISPR-Cas System in CHO Cells

The following sections explore various strategies to enhance recombinant protein (r-protein) production yield in CHO cells using CRISPR technology.

Figure 1. Strategies for Enhancing Recombinant Protein Production in CHO Cells via the CRISPR-Cas System. (Kalkan AK, et al., 2023)

Targeting Apoptosis Genes

Extending the lifespan of CHO cells is crucial for increasing production yield. Disrupting pro-apoptotic genes can significantly reduce the apoptosis rate. For example, knocking down the genes for fucosyltransferase 8 (FUT8), Bcl-2 homologous antagonist/killer (BAK), and Bcl-2-associated X (BAX) all at the same time using CRISPR-Cas9 greatly lowers the pace at which apoptosis is activated and raises the amount of rituximab that is produced. These genes can be knocked off to increase production efficiency while also extending cell longevity.

Targeting Genes that Inhibit r-Protein Yield

Regulating intracellular signaling pathways can increase recombinant protein yield. For instance, the PI3K/AKT/mTOR pathway's inhibition of the TSC2 gene raises mTORC1 activity, which in turn enhances protein synthesis. Similarly, Dnmt3a or Dnmt3b gene knockout increases the long-term production of recombinant proteins considerably by reducing transgene silencing and promoting hypermethylation. Effectively controlling these genes increases CHO cells' ability to produce.

Reducing Metabolic Byproducts

Metabolic byproducts such as ammonia and lactate can inhibit cell growth, affecting protein yield. These byproducts can be decreased by targeting genes involved in lactate production and amino acid metabolism with CRISPR-Cas9. For instance, increasing cell growth rate and protein yield is possible by dramatically reducing lactate production by knocking out the lactate dehydrogenase (LDHA) gene. By optimizing the metabolic pathways in CHO cells, this method effectively raises the efficiency of production.

Customized Production

The production of specific recombinant proteins may require tailored genetic modifications of the host cell line. For example, the production of recombinant human bone morphogenetic protein-4 (rhBMP-4) can be effectively increased by utilizing CRISPR-Cas9 to knock out the BMPRIA or BMPRII genes. By optimizing CHO cell lines for various recombinant protein production requirements, customized genetic editing can greatly increase output.

Gene Knock-In Strategies

Gene knock-in strategies mediated by CRISPR-Cas9 non-homologous end joining (NHEJ) can reduce apoptosis and increase protein yield. For example, integrating the hSurvivin and hQSOX1b genes into CHO cells significantly enhances anti-apoptotic capabilities and recombinant protein production. This gene knock-in strategy not only improves cell survival but also significantly increases protein production.

Gene Regulation Strategies

CRISPR interference (CRISPRi) is another method to increase recombinant protein yield. The dihydrofolate reductase (dhfr) gene's transcription can be inhibited using CRISPR-dCas9, and methotrexate (MTX) selection can be used with this to boost the copy number and expression level of target genes. Furthermore, the CRISPR-Cas13 system, which targets RNA specifically, can enhance the output of recombinant proteins by achieving RNA editing or transcript knockdown. These innovative approaches to gene control offer fresh ways to boost CHO cell production efficiency.

Site-specific integration techniques have transformed CHO cell line creation by utilizing CRISPR-Cas9 and other programmable endonucleases, enabling accurate and effective transgene insertion. These techniques enhance gene expression uniformity, reduce problems related to random integration, and maximize output for biopharmaceutical uses.

For a wide range of research requirements and industrial objectives, Creative Biogene provides complete CRISPR-Cas9 gene editing services. With Creative Biogene's CRISPR technology, you may enhance the production of biopharmaceuticals by efficiently and precisely modifying genes through gene deletion, knockin, or gene regulation. You may create high-yield, high-quality biopharmaceutical products more rapidly and effectively by utilizing Creative Biogene's experience.

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