REPAIRING THE DNA BREAK
A single un-repaired DNA double-strand break (DSB) is lethal to a cell...
but cells have DNA repair capacity. Two primary pathways are utilized to repair DNA DSBs, whether naturally occurring or whether induced by a researcher at a pre-defined genome coordinate (by any homing system that cuts DNA). Breaks occur all the time from molecular mistakes, chemicals or radiation, but in the context of GENE EDITING, the break is both intentional and necessary. The outcomes of each repair pathway are unique and desired depending on research or therapy goals.
NHEJ
NHEJ stands for NON-HOMOLOGOUS END JOINING. It is the typical pathway utilized by a cell in its typical state (G1). During the process, the broken DNA ends are trimmed back and cleaned up by enzymes including Ku70/K80 and Artemis. The DNA is quickly ligated back together, effectively repairing the break. The result is a small deletion of DNA, but this is generally acceptable on a GENOME level, and preferred to cell death.
Researchers use DSBs and the consequences of NHEJ repair to introduce deletions intentionally. Want to knockout a genes function? "NHEJ-it" right in the middle of its exon or coding sequence. One challenge is that not all deletions are equal and clonal cell selection is often desired.
HR and DNA replacement
For replacement gene therapy, HR or HOMOLOGOUS RECOMBINATION is where the magic is. Cells have shown us that when an extra repair "TEMPLATE" is available that cells can use this to fill in the missing information generated by the DNA DSB. For example, right after a cell copies its DNA (Synthesis phase) and before it goes through cell division, there is an extra copy of DNA available. If a break occurs, HR will use this copy to fill in the gap. Highly choreographed and literally using enzymes that temporarily intertwine the 2 DNA strands together, the information from the repair TEMPLATE strand is transmitted into the broken strand.
Researchers now use intentional DNA DSBs, coupled with custom DNA repair templates, to replace or insert a new DNA sequences. The benefits are virtually any sequence can be edited for research and for therapy. The challenges are that HR efficiency is often very low and requires antibiotic resistance selection strategies to select cells with proper repair events. Additionally, for many tissues and cell types in vivo targeting can also be challenging and may require viral vectors. Both represent active areas of research.