Homology directed repair (HDR) is a naturally occurring nucleic acid repair system that can be used to modify genomes in many organisms, including humans. HDR is started by the presence of double strand breaks (DSBs) in DNA. 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.
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.
HDR is a DSBR that uses a double-stranded DNA donor that has homology to the adjacent sequences surrounding the lesion to incorporate new DNA fragments. HDR offers more precision than NHEJ and allows for seamless integration of DNA. The earliest studied HDR pathway is HR. HR uses long, double-strand DNA that is homologous to around 1 kilobase of sequence on either side of the DSB. These homologous DNA donors can come from the sister chromatid, which is used during crossover in meiosis I, or from exogenous sources of DNA like plasmids or PCR products. While HR can be deployed for making exact integrations without indels, it is also inefficient. In cellular models, this can be overcome with selection, like drug resistance or fluorescence. This advantage is lost when attempting to engineer a living animal like a zebrafish, so a more efficient system is required to make knock-ins feasible. It had previously been shown that HDR can be stimulated up to 1000-fold after a DSB in mouse embryonic stem cells, and the boost in efficiency from ZFNs to TALENs was critical for knock-ins in fish.
Genome stability requires the correct and efficient repair of DSBs. In eukaryotic cells, mechanistic repair of DSBs occurs primarily by two pathways: Non-Homologous End-Joining (NHEJ) and Homology Directed Repair (HDR). NHEJ is the canonical homology-independent pathway as it involves the alignment of only one to a few complementary bases at most for the re-ligation of two ends, whereas HDR uses longer stretches of sequence homology to repair DNA lesions.
The HDR process is error-free if the DNA template used for repair is identical to the original DNA sequence at the DSB, or it can introduce specific mutations into the damaged DNA.
There are four different HDR pathways to repair DSBs. Here are only three central steps of the HDR pathways:
- The 5’-ended DNA strand is resected at the break to create a 3’ overhang. This will serve as both a substrate for proteins required for strand invasion and a primer for DNA repair synthesis.
- The invasive strand can then displace one strand of the homologous DNA duplex and pair with the other; this results in the formation of the hybrid DNA referred to as the displacement loop (D loop). This is the defining point of HDR.
- The recombination intermediates can then be resolved to complete the DNA repair process