Hiraizumi, etc. al.
While CRISPR is probably 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 altering specific bases. In all these cases, researchers are trying to balance a number of competing factors: convenience, flexibility, specificity and precision for editors, low error rates, and so on.
So it can be a good thing to have additional editing options, allowing new ways to balance those different needs. On Wednesday, a pair of papers in Nature describe a DNA-based parasite that moves itself around bacterial genomes through a mechanism not previously described. It is far from ready for use in humans, but it may have some distinguishing features that make it worth further development.
Go 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 indeed mobile and appear at new locations in the genome, sometimes by turning themselves off and jumping to new locations, sometimes by sending a copy to a new spot in the genome. To make all this work, they need an enzyme that cuts DNA and specifically recognizes the correct transposon sequence to place in the cut.
The specificity of that interaction, which is necessary to ensure that the system only inserts new copies of itself, and the cutting of DNA, are features that we would like to see in gene editing, which values a better understanding of these systems.
Bacterial genomes typically have very few transposons; the extra DNA is not really consistent with the bacterial reproduction approach of ‘copy all the DNA as quickly as possible when there is food around’. Yet bacterial transposons exist, and a team of scientists from the US and Japan have identified one with a rather unusual feature. As an intermediate step in moving to a new location, 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 nearby DNA (called a “promoter”). When linear, each of the two stretches of DNA on either side of the junction lacks the ability to act as a signal; it only works if the transposon is circular. And the researchers confirmed that there is in fact an RNA produced by the circular shape, even though the RNA does not code for any protein.
So the research team looked at more than 100 different relatives of IS110 and found that they could all produce similar non-protein-coding RNAs, all of which had some key features in common. These include stretches where nearby parts of the RNA could base with each other, leaving an unpaired loop of RNA in between. Two of these loops contained sequences that either base-paired with the transposon itself or at sites within the transposon E.coli genome where it is inserted.
This suggests that the RNA produced by the circular shape of the transposon helped act as a guide, ensuring that the transposon’s DNA was used specifically and inserted only at precise locations in the genome.
Editing without precision
To confirm this was correct, the researchers developed a system in which the transposon would produce a fluorescent protein when properly inserted into the genome. They used this to show that mutations in the loop that recognized the transposon would prevent it from being inserted into the genome – and that it was possible to direct it to new locations in the genome by copying the recognition sequences into the second loop to change.
To demonstrate that this potential was 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 locations in a genome. It could also be used in targeting RNAs that caused the deletion of specific DNA sequences. All of this is potentially very useful for gene editing.
Emphasis on ‘potential’. 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 any sequence of bases to have an insertion site about once every 250 bases.
That relatively low specificity turned out. At the high end, several experiments saw insertion accuracy ranging from an almost usable 94 percent to a positively threatening 50 percent. For deletion experiments, the low end of the range was a catastrophic 32 percent accuracy. So while this has some hallmarks of an interesting gene-editing system, there’s still a lot of work to be done before it can fulfill that 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 that at this point.