The CRISPR-Cas technology enables rapid and precise genome editing at any desired genomic position in almost all cells and organisms. For therapeutic application, it is crucial to bias repair outcomes towards high fidelity homology directed repair (HDR) and to avoid error prone nonhomologous end-joining (NHEJ). In this study, the impact of different repair templates on the frequency of homology directed repair (HDR) and non-homologous end-joining (NHEJ) has been analyzed.A stable HEK293 cell line expressing TLR3 was used to quantify HDR and NHEJ events. 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). Six different CRISPR-Cas9 target sites were identified upstream or downstream of the stop codon within the GFP sequence containing the initiating 5 G and the 3 PAM (NGG).DNA repair activity was measured by FACS.Guide RNAs targeting the active strand (T5, T7) showed higher NHEJ frequencies compared to guide RNAs targeting the inactive strand. HDR activity was highest 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. Circular plasmid was least efficient in generating HDR events.The effect of the different repair templates on NHEJ frequencies was marginal. The results demonstrate the importance of the design of theguide RNA and template DNA on the frequency of DNA repair events and thus, ultimately on the outcome of treatment approach using HDR.
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