Director, Discovery Sciences, R&D
Addressing a key challenge in the production of precise gene-edited models, our recent publication in Nature Communications describes Xential, a novel CRISPR/Cas9-based method to select and enrich for cells with two edited copies of a gene of interest. This approach is already helping us shorten timescales for the development of disease models today, and has the potential to be an indispensable genetic engineering tool in the future.
Finding a needle in the haystack
CRISPR/Cas9-based gene editing has truly revolutionised genetic engineering and allowed us to visualise new scientific frontiers in drug discovery through the creation of cell and in vivo disease models to test new therapeutics. Since its inception less than a decade ago, we, alongside researchers around the world, are continuing to refine CRISPR-based platforms to make it more precise and more functional in the types of gene editing applications that can be achieved.
At AstraZeneca, our growing experience with CRISPR-based tools has allowed us to expand its use across R&D, helping us create new gene-edited disease models to advance drug discovery. This has not been without its challenges, with low editing efficiencies making it hard to select and scale up a relatively small number of cells that carry the desired edits, the proverbial needle, from a larger haystack of “unedited cells”. The current process is labour- and time-intensive and as a result, we have had to devise new approaches to rapidly identify and expand cell populations that have been edited using CRISPR/Cas9 or base editing technology.
Using a bacterial toxin-based selection for precise genome engineering in human cells
Described in the recent Nature Communications publication, we harnessed a novel cell selection method based on the diphtheria toxin receptor. Using CRISPR/Cas9 to introduce a change in both copies (alleles) of the diphtheria toxin receptor gene (HBEGF) makes the cells resistant to the toxin. When diphtheria toxin is applied to a population of cells edited at the HBEGF gene and recruiting any other gene of interest at the same locus, only the cells carrying the edits and the gene of interest in both HBEGF copies are rendered resistant to the toxin, with unedited or partly edited cells being killed by the toxin. This offers a simple and quick approach to enrich the pool of correctly edited cells. As we show in the publication, this system can be applied in vitro, in therapeutically relevant cell types such as human inducible pluripotent stem cells (hiPSC).
The diphtheria toxin selection system has also delivered another powerful advance in the field of CRISPR. CRISPR/Cas9-based gene editing is typically more efficient in producing gene knock-outs, i.e. taking out an incorrect gene or part of a gene, than it is for generating gene knock-ins. Introducing diphtheria toxin-resistant mutations shows an increase in simultaneous, second-site gene editing events in such cells. This increase in gene editing efficiency using Xential has the potential for rapidly co-selecting for a variety of editing events in cells, from changing a single nucleotide, to small insertions and deletions, and even precise large-fragment gene knock-ins. This co-selection system works in primary human T cells, as well as in vivo in mice with a humanised liver.
Overall, Xential offers significant advantages as a simple, universal principle for CRISPR-based gene editing with diverse applications. We are now pushing the boundaries of this novel technology to generate parallel cell pools of disease-associated variants and creating in vivo models of disease that were not previously tractable. Xential is firmly embedded in our in-house target discovery and validation programmes and we are very excited by the prospect of this technology in potentially advancing the development of a broad range of cell and gene therapy applications and the generation of disease models in the future.
Xential has brought down the time it takes to create a new nearly clonal cell pool to just one week, compared with the 4-6 weeks needed via traditional cell enrichment processes.
1. Li et al. Nature Communications 2020. https://www.nature.com/articles/s41467-020-20810-z
Xential is a proprietary technology developed by AstraZeneca, pending patent approval.