Whitepaper Summary:
Recently, several exciting advances in CHO cell line engineering have received significant media and research attention due to efforts in genome sequencing, systems biology, and bioinformatics combined with the relatively new field of targeted gene editing platforms. Three key gene editing technologies have been at the forefront of the recent developments in CHO cell line engineering. Early efforts to introduce targeted site specific edits to the CHO genome focused on implementing the zinc finger nucleases (ZFNs) and the transcription activator-like effector nucleases (TALENs). The ZFN platform has been successfully deployed in a variety of post translational modification applications aimed at increasing specificity of recombinant protein production, but the efficiency of this platform can be limited in mammalian cell lines.
The clustered regularly interspaced short palindromic repeats associated 9 (CRISPR/Cas9) targeted gene editing system has recently exploded onto the research scene in almost every organism. This targeted gene editing platform allows for the creation of multiplexed edits in a single cost-effective step with a specificity previously unachievable in the genome editing arena. This complex is composed of short guide RNAs (sgRNAs) and a CRISPR-RNA that form a site specific construct that is complimentary to the target DNA, which introduces a double stranded DNA break upon binding. Repair of the break site by endogenous enzymes then creates a highly specific change to the DNA which can be customized for a variety of applications.
These advances in genome editing have helped enable high-throughput development of CHO cell lines that can be utilized as economically viable commercial expression vectors. The CRISPR/cas9 gene editing system has shown to be an extremely useful tool for customizing the metabolic pathways of CHO cell lines for use in biopharmaceutical production. One of the most useful applications of this exciting technology has been the creation of multiplexed targeted knock out screening systems. Previously, knock out experiments had to rely on mutagenesis, drug knock out, or media screening to identify the effect of a mutation on a desired cell type. These methods are inefficient and sometimes lead to less desirable off-target effects. It is now possible to develop very large gene knockout libraries to be targeted by CRISPR/cas9 using bioinformatics software specific to this platform.
Customizing metabolic pathways in CHO cell lines is of paramount importance for developing “cell factories” capable of biopharmaceutical production. Reducing the energy expenditure associated with mitochondria production and oxidative metabolism is one approach that has been shown to increase the efficiency of the cell by directing metabolism towards production of the target product. Modulating these pathways has traditionally been accomplished using interfering RNAs, but the specificity of the CRISPR/cas9 platform offers another tool with which researcher can customize the energy profile of CHO cell lines destined for biopharmaceutical production.
Future efforts in the field will be focused on increasing the efficiency of the CRISPR/cas9 system, as this platform is poised to become the model for the majority of biopharmaceutical development. Currently, 60 to 70% of all biopharmaceutical production is accomplished with recombinant mammalian cell lines, and this market share is expected to grow. Multiplexed editing efforts will also continue to increase the rate at which specific customizable CHO cell lines can be produced, as this process is highly critical to increasing the rate at which CHO cell line engineering moves forward.
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