Imma Perfetto
Cosmos science journalist
Scientists have used protein engineering and an AI model to make the bacterial protein TnpB an effective gene editing tool for mammalian cells. TnpB is much smaller than the CRISPR-Cas system and could be more easily delivered to the right cells of the body as a result.
“By engineering the small but powerful protein TnpB, we were able to design a variant that shows a 4.4-fold increase in efficiency of modifying DNA – making it more effective as a gene editing tool,” says Gerald Schwank of the University of Zurich, Switzerland, lead author of the paper in Nature Methods.
The CRISPR-Cas system, which evolved in bacteria and archaea to fight off viruses, has revolutionised genetic engineering since its discovery in 2012.
It consists of an RNA molecule (CRISPR), which finds and locks onto a specific string of DNA, and the CRISPR associated protein, Cas, which makes a cut in the DNA to edit the genetic information there in a precise manner.
It’s a powerful tool, however, the large size of Cas proteins creates challenges when trying to deliver them to the right cells in the body.
But recent research has suggested that Cas proteins evolved from much smaller proteins, with TnpB being the progenitor of Cas12. The only problem is it functions less efficiently, making more unintentional, off-target edits.
To overcome this hurdle, the researchers modified the TnpB protein to better target the DNA of mammalian cells.
“The trick was to modify the tool in two ways: first, so that it more efficiently goes to the nucleus where the genomic DNA is located, and second, so that it also targets alternative genome sequences,” says Kim Marquart, a PhD student in Schwank’s lab and first author of the study.
They also tested TnpB at more than 10,200 DNA target sites to identify which features in the DNA sequences determine the protein’s genome editing efficiency.
They then used this data to develop a new artificial intelligence model capable of predicting how well TnpB will work in different scenarios.
Marquart says this makes it making it easier and faster to design successful gene editing experiments.
“Using these predictions, we achieved up to 75.3% efficiency in mouse livers and 65.9% in mouse brains,” Marquart says.
They showed that TnpB could also be delivered into cells in the live animal models more easily than CSRISPR-Cas.
“For the animal experiments, we were able to use clinically viable Adeno-associated viral vectors to efficiently transport the tools into mouse cells,” says Marquart.
“Due to its small size, the TnpB gene editing system can be packaged into a single virus particle.”
The virus particle acts like a courier to transport the TnpB packaged up within it into target cells. But larger Cas protein components must be packaged into multiple virus particles, meaning higher AAV doses are needed.
With continued concerns about the safely of using high-dose viral therapies in humans, TnpB could be an attractive alternative.
