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Knut Stieger, Fei Song, Birgit Lorenz; OPTIMIZING THE REPAIR TEMPLATE FOR HOMOLOGY DIRECTED REPAIR OF DOUBLE STRAND BREAKS. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4471.
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© ARVO (1962-2015); The Authors (2016-present)
In vivo genome editing takes center stage in the development of therapeutic applications of inherited retinal dystrophies. In such therapeutic applications it is crucial to bias repair outcomes towards high fidelity homology directed repair (HDR) or microhomology mediated end-joining (MMEJ), and to avoid error prone nonhomologous end-joining (NHEJ). In this study, we analyzed the impact of different repair templates on the frequency of HDR and NHEJ in a well-defined cell culture system.
The modified TLR system (TLR3) comprises a bicistronic expression system of a non-functional green fluorescent protein (GFP) gene, followed by a self-cleaving T2A peptide and a second blue fluorescent protein (BFP) gene in a reading frame shifted by 2 bp. A stable HEK293 cell line expressing TLR3 was generated by transfecting a linearized pcDNA3.1(-)-TLR3 plasmid followed by neomycin selection. Donor templates of 1000 bp length containing the corrected GFP sequence were generated as circular plasmid, linearized plasmid with long 3’ or 5’ backbone overhang, or as PCR product. The sequence to be corrected was either centrally located (RS55), with a shorter 5’ homologous region (RS37), or a shorter 3’ homologous region (RS73). Eight different guide RNAs were generated and the Cas9 from the streptococcus pyogenes was used. DNA repair activity was measured by FACS.
Guide RNAs targeting the active strand (T5, T7) showed NHEJ frequencies of about 20%, while guide RNAs targeting the inactive strand showed NHEJ frequencies of less than 5%. HDR activity was highest (about 2%) when using the linearized plasmid with the short 5’ backbone overhang and the RS37 design, followed by the PCR product or the linearized plasmid with the long 5’ backbone overhang, both with RS73 design (1-1.4%). Circular plasmid was least efficient in generating HDR events (<1%). The effect of the different repair templates on NHEJ frequencies was marginal.
We show that a linearized plasmid with short 5’ backbone and short 5’ homology arm is the most efficient variant to induce HDR, proving the importance of the repair template design when developing therapeutic applications for genome editing. However, NHEJ activity is not reduced by different repair templates, meaning that this pathway needs to be repressed by other mechanisms.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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