A fish embryo; the green glow shows that the gene that produces green fluorescent protein has been successfully inserted into the fish DNA. Image credit: Gutierrez-Triana et al. (CC BY 4.0)
CRISPR/Cas9 technology has revolutionized the ability of researchers to edit the DNA of any organism whose genome has already been sequenced. In the editing process, a section of RNA acts as a guide to match up to the location of the target DNA. The enzyme Cas9 then makes a cut in both strands of the DNA at this specific location. New segments of DNA can be introduced to the cell, incorporated into DNA ‘templates’. The cell uses the template to help it to heal the double-strand break, and in doing so adds the new DNA segment into the organism’s genome.
A drawback of CRISPR/Cas9 is that it often introduces multiple copies of the new DNA segment into the genome because the templates can bind to each other before being pasted into place. In addition, some parts of the new DNA segment can be missed off during the editing process. However, most applications of CRISPR/Cas9 – for example, to replace a defective gene with a working version – require exactly one whole copy of the desired DNA to be inserted into the genome.
In order to achieve more accurate CRISPR/Cas9 genome editing, Gutierrez-Triana, Tavhelidse, Thumberger et al. attached additional molecules to the end of the DNA template to shield the DNA from mistakes during editing. The modified template was used to couple a stem cell gene to a reporter that produces a green fluorescent protein into the genome of fish embryos. The fluorescent proteins made it easy to identify when the coupling was successful.
Gutierrez-Triana et al. found that the additional molecules prevented multiple templates from joining together end to end, and ensured the full DNA segment was inserted into the genome. Furthermore, the results of the experiments showed that only one copy of the template was inserted into the DNA of the fish. In the future, the new template will allow DNA to be edited in a more controlled way both in basic research and in therapeutic applications.
