Nuclease based approaches like CRISPR-Cas9, ZFNs and TALENs facilitate targeted modification of genomes by inducing double-strand breaks (DSBs) within chromosomes at specified locations. This stimulates the natural DNA-repair mechanisms of homologous recombination and non-homologous end joining.
Plasmid DNA, PCR products, and single stranded oligonucleotides are routinely used as donors to introduce specific changes at the DSB site. The efficiency of introducing a desired change is dependent on many factors including
- the type of donor
- the length of homology
- the complexity of the desired change
- characteristics specific to the cell line
We have looked at whether rAAV vectors can be used as donors for DNA modification to obtain higher efficiencies than seen with other donor approaches.