Hiraizumi, etc. al.
While CRISPR is perhaps the most prominent gene editing technology, there are others, some developed before and since. And humans have developed CRISPR variants to perform more specialized functions, such as changing specific bases. In all these cases, researchers are trying to balance a number of competing factors: convenience, flexibility, specificity and accuracy for editing, low error rate, etc.
So having additional editing options can be a good thing, enabling new ways to balance these different needs. On Wednesday, a pair of papers in Nature describe a DNA-based parasite that moves itself around bacterial genomes through a previously undescribed mechanism. It is by no means ready for use in humans, but it may have some distinguishing features that make it worth developing further.
Going mobile
Mobile genetic elements, commonly called transposons, are fairly common in many species—they account for nearly half of the sequences in the human genome, for example. They are indeed mobile, appearing in new places throughout the genome, sometimes cutting themselves out and jumping to new places, other times sending a copy to a new place in the genome. For any of these to work, they must have an enzyme that cuts the DNA and specifically recognizes the correct transposon sequence to insert into the cut.
The specificity of that interaction, needed to ensure that the system inserts only new copies of itself, and DNA cutting, are features we would like for gene editing, which adds value to a better understanding of these systems.
Bacterial genomes tend to have very few transposons—the extra DNA is incompatible with the bacterial reproduction approach of “copying all the DNA as fast as possible when there’s food around.” However, bacterial transposons do exist, and a team of scientists based in the US and Japan identified one with a rather unusual feature. As an intermediate step in moving to a new site, the two ends of the transposon (called IS110) are joined together to form a circular piece of DNA.
In its circular form, the DNA sequences at the junction act as a signal that tells the cell to make an RNA copy of the nearby DNA (called a “promoter”). When linear, each of the two pieces of DNA on either side of the junction lacks the ability to act as a signal; only works when the transpose is circular. And the researchers confirmed that there is in fact an RNA produced from the circular form, although the RNA does not code for any protein.
So the research team looked at over 100 different relatives of IS110 and found that they could all produce similar non-protein-coding RNAs, all of which shared some key characteristics. These included stretches where adjacent sections of RNA could pair with each other, leaving an unpaired loop of RNA in between. Two of these rings contained sequences that were either base-paired with the transposon itself or at sites in E. coli the genome where it is inserted.
This suggests that the RNA produced by the circular form of the transposon helped act as a guide, ensuring that the transposon DNA was used specifically and inserted only into the correct locations in the genome.
Editing without precision
To confirm this was correct, the researchers developed a system where the transposon would produce a fluorescent protein when properly inserted into the genome. They used this to show that mutations in the loop that recognizes the transposon would stop it from inserting into the genome—and that it was possible to direct it to new locations in the genome by changing the recognition sequences in the second loop.
To show that this was potentially useful for gene editing, the researchers blocked the transposon’s own RNA production and gave it a functioning replacement RNA. So you could potentially use this system to insert arbitrary DNA sequences into arbitrary locations in a genome. It can also be used to target RNAs that cause the deletion of specific DNA sequences. All of this is potentially very useful for gene editing.
Emphasis on “potentially”. The problem is that the target sequences in the loops are quite short, with the insertion site targeted by a recognition sequence that is only four to seven bases long. At the short end of this range, you would expect a random sequence of bases to have an insertion site about once every 250 bases.
This relatively low specificity showed. At the highest level, various experiments could see insertion accuracy ranging from a near-helpful 94 percent to a menacingly positive 50 percent. For sweep experiments, the low end of the range was a disastrous 32 percent accuracy. So while this has some of the makings of an interesting gene editing system, it has a lot of work to do before it fulfills this potential. It’s possible that these recognition loops could be made longer to add the kind of specificity that would be needed for editing vertebrate genomes, but we just don’t know at this point.