If you are a scientist working in molecular biology, hardly does a week go by without reading another exciting publication on genome editing. The majority of the studies are utilising the CRISPR/Cas9 system that has been made available through reagent depositories and as such dozens of studies each month elucidate the feasibility of precise genome editing in a number of organs and in multiple species.
Whether it is for disease modelling applications or therapeutic ones, in vitro studies in cultured cells or direct in vivo administration into organisms, genome editing has been used for both inactivation of genes to study their function or precise correction/addition of a gene sequence in a specific genomic position.
The former mechanism usually requires just to cleave the DNA within a gene and then allowing the cell to imperfectly “stitch” the DNA together. As the cell doesn’t like “loose ends”, it goes into a kind of panic mode when this happens adding “spelling mistakes” within the code that will cause that gene to stop working. This is a useful and quick way to study the function of a gene. It’s called non-homologous end joining (NHEJ).
The latter mechanism of precise genomic alteration requires the presence of a template so that the cell can use it to perform homologous-directed repair (HDR) and insert it in its chromosomes without the addition of mutations. This is a more laborious method but extremely useful when in need to correct a disease-causing mutation or insert a reporter gene without disrupting the function of the endogenous one.
HDR up to now has been thought to be very inefficient in postmitotic cells, i.e. cells that do not divide, and this has been hampering the efforts to perform meaningful gene correction in most organs. The majority of the cells in our body are postmitotic and while HDR has been used extensively in induced pluripotent stem cells (iPS) to study and correct defects in development, in vivo HDR has been elusive.
In this study by Nishiyama and colleagues (Nishiyama et al. 2017), the authors used Adeno-associated viruses (AAVs) to deliver both the Cas9 nuclease and its guide molecule together with a template for precise HDR within genes, in the (postmitotic) brain of rodents. While it’s known that AAV vectors due to their genomic structure and configuration can stimulate the cell to undergo HDR instead of NHEJ, the extent to facilitate meaningful in vivo HDR has not been shown. Here, localised injection in the brain led to HDR in neurons that reached double digits in percentage of cells, something that was previously thought to be extremely unlikely. The exact mechanism by which AAVs are able to induce this shift towards HDR in postmitotic cells is not known. The authors demonstrated that such applications are feasible in both healthy and diseased models (a mouse model for Alzheimer’s disease was also used).
While it is noted that the efficacy of HDR most probably varies between cell types in the body as well as being affected by the position of the target sequence within the genome, this study will most likely pave the way for a number of future studies that will test this mechanism in a variety of tissues. The technique might need optimisation to increase the levels of HDR as seen here in the central nervous system and even then depending on the nature of the disease we aim to treat, the levels of HDR might not be enough to elicit a therapeutic effect for many of them. However, a number of disorders might be amenable to partial or full treatment that was previously not possible.
So watch this space as the panoply of genome editing applications just got bigger and its growth doesn’t seem to be slowing down at all!
Nishiyama, J., et al. (2017). “Virus-Mediated Genome Editing via Homology-Directed Repair in Mitotic and Postmitotic Cells in Mammalian Brain.” Neuron.