Eric Rhodes Chief Technology Officer Horizon Discovery

Genome editing has gotten a real boost from the recent interest in CRISPR technology. The majority of CRISPR papers being published utilize the technology to functionally disrupt (knock-out) a gene using an NHEJ based mis-repair system. Using CRISPR to purposefully edit the genome in a more controlled manner is a slightly more challenging proposition that calls upon the endogenous homology directed repair system and requires one additional component not needed in knock-out experiments… a “donor” DNA template.

For the most part, researchers have been using either single-stranded oligos or double-stranded plasmids to provide this homology template. Because of our long history of having used rAAV (recombinant adeno-associated virus) to edit and create hundreds of isogenic lines, Horizon was curious to see whether the combination of rAAV with a CRISPR-induced break might lead to levels of targeted recombination higher than those seen with either an oligo or a double-stranded plasmid.

rAAV has been known for some time to be able to drive levels of homologous recombination that are up to 1,000-fold higher than those seen when using a simple plasmid alone. Despite this vast improvement over the use of plasmids, the levels of recombination seen are still relatively modest using rAAV alone, but have been seen to be boosted significantly when a double-strand break is introduced in the vicinity of the homology region.

We used CRISPR to introduce double-strand breaks at various points in the genome of a cell line (HCT116) carrying a disabled copy of the GFP gene on one chromosome. The figure below shows the organization of the artificial GFP gene split into three exons, with the third exon carrying a mutation, which prematurely terminates the GFP protein rendering it non-functional. Conversion of the A residue to a C restores GFP functionality. The location and relative distance of five different gRNA targets sites from the point mutation are shown below the expanded Exon 3 region.

We used both a plasmid and rAAV derived from that same plasmid as a donor and measured the ability of these donors to stimulate a genetic modification at a range of distances from the induced cut site. 

Remarkably the AAV donor performs markedly better than the corresponding plasmid with an average 10-fold improvement at most distances. We are working to extend this work and recent results comparing rAAV with oligo and plasmid donors at different concentrations have supported this trend.

CRISPR has already proven itself to be an incredibly useful tool for disruption of multiple genes. We feel however, that the true power of gene editing and the promise of CRISPR will shine when researchers can routinely recreate the full variety of patient-specific SNPs, which may be contributing to disease or resistance to treatments. CRISPR alone may not be sufficient to fully realize this dream, but through combinations of technologies, like rAAV-assisted CRISPR genome editing, we hope to take researchers one step closer to this reality.

Eric Rhodes ([email protected]) is SVP, R&D and CTO of Horizon Discovery.

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