Hiraizumi, et al. al.
While CRISPR is probably the most prominent gene editing technology, there are​​​​​​ others, some developed earlier and later. And people have been developing CRISPR variants to perform more specialized functions, such as changing specific bases. In all these cases, researchers try to balance a number of competing factors: convenience, flexibility, specificity and accuracy of adjustments, low error rate, and so on.
So having more customization options can be a good thing, allowing for new ways to balance these different needs. On Wednesday, two papers in Nature describe a DNA-based parasite that moves around bacterial genomes by a previously undescribed mechanism. It is far from ready for use in humans, but it may have some characteristics that make it worth further development.
Switch to mobile
Mobile genetic elements, commonly called transposons, are quite common in many species – for example, they make up almost half of the sequences in the human genome. They are truly mobile, appearing in new locations throughout the genome, sometimes by cutting off and jumping to new locations, other times by sending a copy to a new location in the genome. For any of this to work, they need to have an enzyme that cuts the DNA and specifically recognizes the correct transposon sequence to insert into the cut.
The specificity of this interaction, needed to ensure that the system only inserts new copies of itself, and the cutting of DNA are features we would like for gene editing, which emphasizes a better understanding of these systems.
Bacterial genomes tend to have very few transposons—the extra DNA isn’t really consistent with the “copy all the DNA as fast as you can when there’s food around” approach to bacterial reproduction. However, bacterial transposons do exist, and a team of scientists based in the US and Japan has identified one with a somewhat unusual feature. As an intermediate step in moving to the 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 sequence at the junction acts as a signal that tells the cell to make a copy of the RNA near the DNA (referred to as the “promoter”). When linear, each of the two pieces of DNA on either side of the junction lacks the ability to function as a signal; it only works when the transposon is circular. And scientists have confirmed that there is in fact RNA produced in a circular form, although RNA does not code for any proteins.
So the research team looked at more than 100 different relatives of IS110 and found that they can all produce similar non-coding RNAs, all of which share some key properties. These included stretches where nearby stretches of RNA could pair with each other, leaving an unpaired loop of RNA in between. Two of these loops contained sequences that were either paired with the transposon itself or at sites v E-coli of the genome where it was 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 specifically used and inserted only into precise locations in the genome.
Editing without precision
To confirm that this was correct, the researchers developed a system where the transposon would produce a fluorescent protein when inserted correctly into the genome. They used this to show that a mutation in the loop that recognizes the transposon prevents its insertion into the genome—and that it is possible to direct it to new locations in the genome by changing the recognition sequences in the other loop.
To show that it was potentially useful for gene editing, the researchers blocked the production of the transposon’s own RNA and fed it a replacement RNA that worked. So you could potentially use this system to insert arbitrary DNA sequences into arbitrary places in the genome. It could also be used with targeted RNAs that caused the removal of specific DNA sequences. All of this is potentially very useful for gene editing.
Emphasis on “potentially”. The problem is that the targeting sequences in the loops are quite short, with the insertion site being 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 string of bases to have an insertion site about once every 250 bases.
It turned out to have relatively low specificity. At the highest level, various experiments could see insertion accuracy ranging from a near-useful 94 percent to a positively terrifying 50 percent. For the deletion experiments, the lower end of the range was a disastrous 32 percent accuracy. So while it has some features of an interesting gene editing system, there is still a lot of work to be done before it can fulfill its potential. It’s possible that these recognition loops could be longer to add some of the specificity that would be needed to edit vertebrate genomes, but we just don’t know at this point.