Scientists have created a new version of the CRISPR/Cas9 genome editing technology that allows them to activate genes without creating breaks in the DNA, potentially circumventing a major hurdle to using it to treat human diseases.
Most CRISPR/Cas9 systems work by creating "double-strand breaks" (DSBs) in regions of the genome targeted for editing or for deletion, but many researchers are opposed to creating such breaks in the DNA of living humans.
Researchers at Salk Institute for Biological Studies in the US used the new approach to treat several diseases, including diabetes, acute kidney disease, and muscular dystrophy, in mouse models.
"Although many studies have demonstrated that CRISPR/Cas9 can be applied as a powerful tool for gene therapy, there are growing concerns regarding unwanted mutations generated by the double-strand breaks through this technology," said Juan Carlos Izpisua Belmonte, a professor at Salk.
"We were able to get around that concern," said Izpisua Belmonte, senior author of the study published in the journal Cell.
In the original CRISPR/Cas9 system, the enzyme Cas9 is coupled with guide RNAs that target it to the right spot in the genome to create DSBs.
Recently, some researchers have started using a "dead" form of Cas9 (dCas9), which can still target specific places in the genome, but no longer cuts DNA.
The dCas9 has been coupled with transcriptional activation domains - molecular switches - that turn on targeted genes.
However, the resulting protein - dCas9 attached to the activator switches - is too large and bulky to fit into the vehicle typically used to deliver these kinds of therapies to cells in living organisms, namely adeno-associated viruses (AAVs).
The lack of an efficient delivery system makes it very difficult to use this tool in clinical applications.
Izpisua Belmonte's team combined Cas9/dCas9 with a range of different activator switches to uncover a combination that worked even when the proteins were not fused to one another.
In other words, Cas9 or dCas9 was packaged into one AAV, and the switches and guide RNAs were packaged into another.
They also optimised the guide RNAs to make sure all the pieces ended up at the desired place in the genome, and that the targeted gene was strongly activated.
"The components all work together in the organism to influence endogenous genes," said Hsin-Kai Liao, a staff researcher in the Izpisua Belmonte lab.
The technology operates epigenetically, meaning it influences gene activity without changing the DNA sequence.
To test the method, the researchers used mouse models of acute kidney injury, type 1 diabetes and a form of muscular dystrophy.
In each case, they engineered their CRISPR/Cas9 system to boost the expression of an endogenous gene that could potentially reverse disease symptoms.
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