"Mini" CRISPR tool reworks inactive protein for better gene editing
The CRISPR gene-editing system is usually associated with the Cas9 protein, but that’s not the only option out there. Scientists at Stanford have now developed a CRISPR tool called CasMINI, using a much smaller protein that should be able to enter human cells more easily.
CRISPR is a powerful technique for making precise genetic edits in living cells. The tool uses enzymes to cut out targeted sequences of DNA – such as those linked to disease, for example – and replace them with something more useful. This method has shown incredible promise over the last decade or so in potentially treating a wide range of genetic diseases, as well as improving crops and livestock, controlling pests, or manipulating microbes.
The most commonly used enzyme is Cas9, but others have shown promise, too, such as Cas12a, Cas12b, Cas3, and CasX. The problem is that their efficiency is somewhat restricted by their relatively large size, which makes it harder for the machinery to squeeze into cells and get to work. So for the new study, the Stanford team set out to design a miniature CRISPR system.
The team’s new CasMINI is made up of only 529 amino acids, making it less than half the size of other Cas enzymes, which typically have between 1,000 and 1,500 amino acids. Even the previous smallest, CasX, still has almost 1,000.
Of course, size doesn’t matter if your new system doesn’t work as well. But when the team tested it out in mammalian cells in the lab, it was found to be just as effective at deleting, activating and editing DNA as the other Cas enzymes, while having an easier time getting into cells.
But perhaps most impressive about CasMINI is that it was modified from a non-working natural protein called Cas12f. It was an intriguing starting point for making a smaller CRISPR enzyme since it only has between 400 and 700 amino acids, but previous tests showed no activity in human cells. That’s most likely because it originated in microbes called archaea, which have far simpler genomes than humans.
So the Stanford team examined the structure of this enzyme, checking 40 different mutations that could make it more effective while retaining its small size. And sure enough, after several years of iterating, they ended up with a version that worked well.
The team says that this development could not only help make CRISPR more effective, but also might lead to new ways to shrink RNA systems as well, such as those used to develop the mRNA vaccines for COVID-19.
The research was published in the journal Molecular Cell.
Source: Stanford University
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