Basil Hubbard, Canada Research Chair in Molecular Therapeutics and an assistant professor in the U of A’s Department of Pharmacology, and his team have filed a patent on their discovery and are hoping to partner with the pharmaceutical industry to incorporate it into a therapeutic.
Interest in gene-editing technology has been rapidly rising since the discovery of CRISPR/Cas9. This system is naturally present in bacteria, which use it for protection against their natural predators, called bacteriophages.
“It allows bacteria to store information about previous infections and then use it to seek out and destroy the DNA of new invaders by cutting it,” explained Hubbard.
Using its natural RNA guide molecule, the Cas9 system is quite accurate, only making a mistake about one per cent of the time, he noted.
“However, given that there are trillions of cells in the human body, even one percentage off is quite significant, especially because gene editing is permanent. One wrong cut and a patient could end up with a serious condition like cancer.”
The new BNA guide molecule that Hubbard and his team—which includes PhD student Christopher Cromwell, who is first author on the study—developed was shown to be much more stable and stringent in its quest for finding the right DNA to cut.
“Our research shows that the use of bridged nucleic acids to guide Cas9 can improve its specificity by over 10,000 times in certain instances—a dramatic improvement,” said Hubbard.
Though gene-editing technology still has several hurdles to overcome, including the challenge of how to deliver it effectively into the human body, it may someday be used to treat a wide variety of genetic diseases, from muscular dystrophy to hemophilia and various cancers.
Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2′,4′-BNANC[N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNANC incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA–DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, “zipped” conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids.
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