Genome editing has had a real boost recently through the interest in CRISPR technology. However, whilst the majority of CRISPR papers being published utilize the technology to functionally disrupt (knockout) 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 knockout 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. Horizon has a long history of using rAAV (recombinant adeno-associated virus) to edit and create hundreds of isogenic lines, and we were therefore curious to see whether the combination of rAAV with a CRISPR-induced break might lead to levels of targeted recombination higher than seen with either an oligo or a double-stranded plasmid in combination with CRISPR.
It has been known for some time that rAAV is able to drive levels of homologous recombination that are up to 1,000-fold higher than when using a simple plasmid. Despite this vast improvement compared with plasmids, the levels of recombination seen are still relatively modest using rAAV alone. Recombination rates have however been seen to be boosted significantly when a double-strand break is introduced in the vicinity of the homology region.
To investigate the effect of combining CRISPR and rAAV, 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. Figure 1 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 nonfunctional. 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.