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.
Creation of a gene targeting efficiency reporter system
To measure rates of HR and targeting frequencies, we generated cell lines with an integrated GFP reporter gene inactivated by a nonsense mutation to prevent fluorescence. Donor molecules could then be used to integrate the correct sequence to revert the mutation and switch-on GFP, enabling rates of HR to be measured by flow cytometry
We refer to this as the Fluorescent Indicator of Recombination efficiency line, or FIRE-Line for short.
An overview of FIRE-Line locus and gRNAs targeting the FIRE-Line locus. Schematic representation of the FIRE-Line locus (Top) constituting an inactive split GFP driven by CMV promoter integrated into the HPRT locus of HCT116 (Middle). The location of the gRNAs used in the experiment are shown (Bottom).
Donors design for testing gene editing efficiency
ssODN, dsDNA plasmid, and rAAV donor. Constructed to restore GFP activity upon targeted incorporation.
Briefly, donors encode a correction for functional GFP when recombined in correctly. Cells were transfected with Cas9 + guide RNA along with each separate donor and on day 6 the transfected cells were analysed by FACS to measure the proportion of correctly targeted cells.
Summary of gene-targeting frequencies at the FIRE-Line locus. The targeting efficiency of three donor formats was measured in the HCT116 FIRE-Line locus. rAAV gave higher targeting frequencies with both wild type Cas9 wt (Blue) and Cas9 nickase (Red) compared to a ssODN or plasmid donor.
The proportion of correctly targeted cells increases by 30-100 fold when rAAV is used as a donor compared to the other donors. This significant improvement in targeting efficiency stands to impact gene editing workflows by:
- Reducing the number of clones to be screened to identify correctly targeted clones.
- Significantly reducing time and cost for creating a cell line.
- Making hard to transfect cells feasible backgrounds for gene editing projects
Got a challenging gene editing project?
Along with our above ability to combine gene editing tools for maximum efficiency, Horizon has unmatched expertist in gene editing - to date we’ve completed over 2,000 genome editing projects in more than 70 different cell lines, often on extremely tight deadlines.
So if you're research requires a genetically engineered cell line model we'd love to help!