CRISPR, which stands for ‘clustered regularly interspaced short palindromic repeat’ was first detected in E. coli in 1987. Most notably, however, its interaction with the endonuclease Cas9, along with the recognition that this peculiarity of the prokaryotic immune system may be used to accurately and permanently modify the genetic code in mammalian cells, was not discovered until many years later (Charpentier et al. 2012, Mali et al. 2012).
This was the beginning of what is often considered to be the gene-editing revolution.
Since that time, CRISPR/Cas9-based gene editing has become a cornerstone technology in the genetic manipulation of cells; both in commercial and academic laboratories.
This technology has huge potential and is already beginning to impact the whole scientific spectrum ranging from drug discovery through to direct therapeutic applications. However, like any pioneering technology, it is not without its issues.
Generating a CRISPR-modified cell that has been grown from a single-cell clone is a time-consuming process. It is also inefficient, often being carried out by hand, which leaves it prone to errors. These limitations adversely impact on the clinical or commercial scalability of this technology.
The scale is not the only issue. Questions remain around the accuracy and efficiency of CRISPR/Cas9 for genome editing.
The CRISPR system is fairly straightforward, despite its considerable impact on biotechnology. A guide RNA directs a Cas9 protein towards a DNA sequence that is complementary to the RNA.
Next, the Cas9 cuts, establishing a double-strand break (DSB). The cell is then able to repair the cut through either non-homologous end joining, which commonly results in knock-out mutations, or homology-directed repair, which can be employed to knock-in mutations.
Regardless of which repair method is used, this conventional style of CRISPR/Cas9 editing is heavily reliant on the guide RNA being able to direct the Cas9 solely to the specific target site. If Cas9 binds non-specifically, off-target mutations may arise, prompting unwelcome genome modifications.
Gene editing advances have aimed to enhance the accuracy of gene editing. Base editing requires elements of the CRISPR/Cas9 system but works alongside other enzymes to directly insert specific point mutations in the DNA of nondividing cells, avoiding the creation of double-strand breaks (Komor et al. 2016). This substantially lowers the number of unintentional edits.
Prime editing has become a highly regarded technique, particularly by the world’s media. This process involves using a form of Cas9 that nicks the DNA instead of making a full double-strand break.
Rather than providing a guide RNA to target the molecular cut with a new template DNA to interpret the new genetic code, the guide RNA in prime editing does both simultaneously. The DNA nick prompts reverse transcription of the edited guide RNA into the DNA target site, resulting in considerably less off-target mutations (Anzalone et al. 2019).
Neither of these innovations has been able to address the problem of appropriate scaling of this technology, however. But, this is where OXGENE™ excels.
OXGENE’s highly optimized, quality assured automated CRISPR cell line engineering workflow has been assembled on the foundations of a robust multidisciplinary framework.
This combines skills and knowledge of informatics, biology, and automation, utilizing this combination to create an efficient, reliable, and high-throughput genetic engineering platform that consistently edits hundreds of cell lines every year while maintaining impressive pass rates of KO success at the protein level when based on genotype predictions.
OXGENE’s platform undertakes the majority of the repetitive, routine, or time-consuming elements of gene editing automatically. It is these parts of the protocol which are most at risk of human error.
First we automated the process of scanning plates at high throughput. Then we built the IT infrastructure and procedures to deal with clone verification. The next challenge was getting the clone-picking properly automated. But the best bit was building and optimizing the user interfaces so the scientists can be completely in control of their own work, without needing input from the automation team.
Simon Pollack, Group Leader of Laboratory Automation
Pollack thus outlined how OXGENE began building the platform’s infrastructure.
The triumph of OXGENE’s capabilities in the genome engineering industry does not rest exclusively on their robots, however.
OXGENE’s platform is comprised of several essential aspects, not least the solid partnerships between OXGENE’s automation experts, its in-house bioinformatics team who are responsible designing precise primers and guides, biologists responsible for managing the protocols, and its project managers working diligently with various teams to make certain that operational planning and resourcing are effective and efficient.
Despite their successes, OXGENE continues to innovate.
We’re currently optimizing conditions for more complex cell types, like primary cells and stem cells on our high-throughput platform, and also for more complex modifications, such as knock-ins.
Pela Derizioti, Gene Editing Team, OXGENE
Simon sums up OXGENE’s ethos, “Within the year, we’ll have doubled our capacity again.” Pela concludes, “We’ll never finish innovating,”
Produced from materials originally authored by Sophie Lutter from OXGENE.
OXGENE™ combines precision engineering and breakthrough science with advanced robotics and bioinformatics to accelerate the rational design, discovery and manufacture of cell and gene therapies across three core areas: gene therapy, gene editing and antibody therapeutics.
Gene therapy: We’re transforming the vision of truly scalable gene therapies into a reality; progressing our industry leading transient gene therapy systems towards alternative technologies for scalable, stable manufacturing solutions.
Gene editing: We have automated gene editing to deliver CRISPR engineered cell lines at unparalleled speed, scale and quality and generate complex disease models in mammalian cells.
Antibody therapeutics: We’re employing a novel proprietary mammalian display technology to discover antibodies against previously intractable membrane proteins.
OXGENE™ works at the edge of impossible in mammalian cell engineering. Our scientific expertise and technology solutions address industry bottlenecks. For more information, please visit www.oxgene.com
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