DNA containing double-strand breaks (DSB) created by the CRISPR/Cas9 system can be repaired by either the non-homologous end-joining (NHEJ) or the homology-directed repair (HDR) pathway. The NHEJ repair pathway introduces non-specific insertions or deletions at the cleavage site, whereas the HDR pathway allows for precise gene editing at the DSB site. Target-specific HDR Plasmids provide a DNA repair template for a DSB and, when co-transfected with CRISPR/Cas9 KO Plasmids, enable the insertion of specific selection markers where Cas9-induced DNA cleavage has occurred. The HDR plasmid can incorporate a Red Fluorescent Protein (RFP) gene to visually confirm transfection and an antibiotic resistance gene (puromycin) for selection of cells containing a successful CRISPR/Cas9 doublestrand break. The puromycin resistance and RFP encoding genes are flanked by two LoxP sites that are recognized by the Cre Vector, which can be used to later remove these selection markers from the genomic DNA.
