DNA repair mechanisms have evolved into two major types of repairs:14
- Non-homologous end joining (NHEJ): This repair typically occurs after CRISPR/Cas9 cleaves the targeted DNA segment, leaving blunt ends on both strands. The damaged DNA drives the cell-autonomous DNA repair system to immediate action to save the cell. NHEJ repair pieces, however, are not always as exquisite as expected, leading to tiny but undesired insertions or deletions at sites of DNA repair. Either insertion or deletion alters the original DNA sequence, setting the stage for the encoded gene to cease function. This type of inadvertent DNA repair error, as it turns out, is precisely why CRISPR molecular scientists design CRISPR experiments to take place—with an aim at allowing cellular DNA repair system to run its course of rendering the target gene to be silenced or target gene “knock-out.” The practical utility of gene silencing is to reveal the function(s) of the gene when it is fully functional through loss-of-function studies.
- Homology-directed repair (HDR): Since the default cellular DNA repair machinery is NHEJ, HDR must be induced. How do we direct cells to activate this HDR pathway? By providing a slice of donor DNA that harbors DNA ends homologous to the ends at the CRISPR/Cas9 DNA break site. The presence of the homologous donor DNA drives the HDR repair, leading to donor DNA insertion to the site, effectively filling in the gap left by CRISPR/Cas9. This type of gene editing method is termed a “knock-in” study.
Both knock-out and knock-in approaches are useful mechanisms of the CRISPR/Cas9 gene-editing system. Recent approaches and investigations have demonstrated additional CRISPR molecular scissors beyond Cas9 and harbor comparative advantages in DNA break and gene editing capabilities.
14. Reuven, N., & Shaul, Y. (2022). Selecting for CRISPR-Edited Knock-In Cells. International journal of molecular sciences, 23(19), 11919. https://doi.org/10.3390/ijms231911919
