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Using Cas9-induced DNA double-strand cleavage to insert a gene via Homology Directed Repair. 1: Cleavage of desired strand by Cas9 RNA guided nuclease. Addition of a desired insert DNA sequence flanked by regions homologous to each side of cut-site 2: Endogenous Homology Direct Repair mechanism uses homologous regions to rejoin cleaved DNA, this process will occur in both Eukaryotes and Prokaryotes. 3: creation of the intended modified DNA (original figure)
Homology directed repair (HDR), a naturally occurring nucleic acid repair system, can be used to modify genomes in many organisms, including humans (Sander and Joung, 2014). HDR is initiated by the presence of double strand breaks (DSBs) in DNA (Liang et al. 1998). Because the CRISPR/Cas9 system can be used to create targeted double strand breaks, researchers have begun using CRISPR/Cas9 to control the specificity of HDR genome engineering techniques (Findlay et al. 2014; Mali et al. February 2014; Ran et al. 2013).
Following the RNA-guided cleavage of a specific site of DNA, the target cell will also be given large quantities of a donor template. This donor template has the desired insertion or modification, flanked by segments of DNA homologous to the blunt ends of the cleaved DNA. Thus the natural DNA-repair mechanisms of the cell can be used to insert the desired genetic material, editing the genome of a target cell with high-precision. Genome modification carried out in this way can be used to insert novel genes, or knock out existing genes (Mali et al. Feb 2013).
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