The CRISPR gene-editing system is a powerful tool that looks set to revolutionize the way we treat diseases, as well as basically anything else that can benefit from precisely manipulating DNA. The problem is that sometimes cutting and pasting can have unwanted side effects. Now researchers from MIT and Harvard have developed a new CRISPR-based system that can insert new DNA sequences without needing to make cuts, which should make the process safer and more accurate.
Normally, the CRISPR tools are based on a bacterial self-defense system. When the bugs encounter predatory viruses called bacteriophages, they use enzymes like Cas9 to snip a piece of that virus's DNA and store it within themselves. That works a bit like a "Wanted" poster, letting the bacteria easily identify (and fight) the virus if they ever cross paths again.
In the last few years, scientists figured out how to use this technique to make cut-and-paste DNA edits in the cells of other organisms. Guide RNA sequences tell the enzymes where to cut, removing pieces of DNA from the cell and replacing them with new sections. That can be used to correct mutations that cause disease, or even prevent them entirely by snipping the offending sequences out of the genome of an embryo. Along with treating disease in humans, the method could be used in pest control or to make crops hardier and more nutritious.
As useful as CRISPR is so far, it's not without issues. Relying on the cell's natural repair mechanism means that sometimes errors occur, and other times off-target edits may appear in other parts of the genome. That's led researchers to develop other similar methods that may be a bit more gentle, silencing genes instead of cutting them out, or working more like a word processor's "search and replace" function.
The researchers on the new study set out to create a new system with a similarly light touch. Rather than using defensive enzymes like Cas9, the team investigated DNA sequences called transposons. These are also called "jumping genes" thanks to their tendency to jump around in the genome, with some subtypes guided by proteins called transposases.
The team isolated the enzyme Cas12k from two species of cyanobacteria and manipulated them into jumping to set targets in the genome, then inserting new DNA sequences without having to cut anything. The new system was named CRISPR-associated transposase (CAST).
"We dove deeply into this system in cyanobacteria, began taking CAST apart to understand all of its components, and discovered this novel biological function," says Jonathan Strecker, first author of the study. "CRISPR-based tools are often DNA-cutting tools, and they're very efficient at disrupting genes. In contrast, CAST is naturally set up to integrate genes. To our knowledge, it's the first system of this kind that has been characterized and manipulated."
The team tested the new system on E. coli, and were able to insert new DNA sequences up to 10 base pairs long into precise locations on the genome. It worked 80 percent of the time, but that could be improved with further research.
The CAST technique could be used for a few purposes. Genetic diseases could be healed by disabling the harmful mutation and inserting a new, healthy version instead. Or it could add a brand new, useful sequence without silencing any existing gene, effectively inserting a new ability – for example, the immune system's T cells could be targeted to make them more responsive to the presence of cancer.
"For any situation where people want to insert DNA, CAST could be a much more attractive approach," says Feng Zhang, senior author of the study and inventor of the CRISPR system. "This just underscores how diverse nature can be and how many unexpected features we have yet to find."
The research was published in the journal Science.
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