Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing.

Double-strand breaks (DSBs) in DNA occur naturally in every organism (microoorganisms, plants, animals, humans) and are mended by the cell’s own repair mechanisms. The original state before the DNA damage can be restored or the genome can be changed at that particular region, i.e. the introduction of small insertions, deletions or point mutations. In addition – and this is what this study is all about – larger DNA fragments can be incorporated into the area of the DSBs of the genome.
In the study, the DNA sequences of the target region in mouse cells were examined after application of CRISPR/Cas9. In some cases unintentionally foreign DNA fragments had been incorporated at the DNA DSBs. By using the gene scissors CRISPR/Cas9, DSBs are introduced at certain target regions of the genome. Cell-specific repair mechanisms can, as with naturally occurring DNA DSBs, cause small changes in the target region. The majority of the gained changes are small changes at the target sequence. In significantly fewer cases (4%), large DNA fragments were incorporated into the target sequence. The scientists examined these DNA fragments more closely and found that they came from different genomic sources: A large part comes from the vector plasmid with which the DNA of the CRISPR/Cas9 gene scissors was introduced into the cells. A smaller proportion comes from the bacterium E. coli, with which the DNA of the CRISPR/Cas9 gene scissors was duplicated in the laboratory before the gene scissors were then introduced into the cells. Some of the DNA fragments come from the genome of the mouse. Surprisingly, a small proportion of the inserted DNA fragments comes from the genome of cattle. The explanation for this: When working with mouse cells in cell culture, bovine serum must be added to the cells’ nutrient medium so that the cells can multiply and survive. The DNA from cattle contaminates the nutrient medium, so to speak, and is introduced via so-called exosomes, small vesicles that transport small molecules between cells and their environment. The scientists repeated the experiment with goat serum in the nutrient medium of the mouse cells with a similar result: In the target area of the CRISPR/Cas9 application, DNA from the vector plasmid, from E. coli, from the mouse and, to a small extent, DNA from goat were found.
The authors state their findings „represents a potential new risk for genome editing“. As such, the results also raise important aspects for applications of CRISPR/Cas for the risk assessment: In theory, such DNA fragments can also be incorporated in possible off-target areas and naturally occurring DSBs. Off-target areas are locations in the genome that are very similar to the target region and in which the CRISPR/Cas9 gene scissors can unintentionally cause unwanted changes in the genome.
DNA DSBs promote the incorporation of foreign DNA, which is a general problem in the basic concept of targeted nucleases such as CRISPR/Cas. Current research is working on alternative CRISPR/Cas systems that do not cause DSBs in the DNA, such as base editing or prime editing. But also these techniques can cause specific, unintended changes in the genome.

Ono R, Yasuhiko Y, Aisaki K-i, Kitajima S, Kanno J, Hirabayashi Y (2019) Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing. Communications Biology 2 (1):57. doi:10.1038/s42003-019-0300-2