Scientists Improve the Accuracy of CRISPR with Synthetic gRNA

Scientists Improve the Accuracy of CRISPR with Synthetic gRNA

CRISPR-Cas9 allows researchers to make precise edits to DNA to remove defective genes. While the technique can be used to make cuts at specific locations with little off-target effects, researchers at the University of Alberta have developed a new technique that improves the accuracy further still.

One of the main fears of using CRISPR-Cas9 is off-target DNA cleavage. While a defective gene can be removed to cure a genetic disease, off-target cuts could result in considerable harm. Many researchers consider CRISPR-Cas9 to be safe, but not everyone agrees. It is fears over the safety of the gene editing technique which is holding back clinical trials and patient applications.

However, use of the technology could be accelerated thanks to the work of the University of Alberta researchers, who claim their technique reduces the risk associated with CRISPR-Cas9 by ‘several orders of magnitude’.

The CRISPR system is present in bacteria and is used to remove sections of DNA that been altered by bacteriophages. The system has been developed for use in humans, but instead of removing foreign DNA, it can be used to remove and replace faulty genes.

CRISPR uses guide RNA (gRNA) to identify a specific sequence of base-pairs which allows cuts to be made in very specific locations. While accurate, there is around a one in a hundred chance of a mistake being made.

While those odds may seem reasonable, especially for patients suffering from terminal diseases, the problem is there are trillions of cells in the human body. If a mistake is made 1% of the time, the negative effects could be considerable. Since any off-target effects would involve permanent changes to the DNA, it is possible that they could trigger another condition that could be worse – or as bad – as the one the system was used to cure.

Several research teams have been working on CRISPR-Cas9 and have attempted to alter the Cas9 component, which combined with the gRNA is responsible for making the cuts, but there has been little research completed on changing the gRNA that tells the enzyme where the cut should be made.

The solution developed by the University of Alberta researchers involves replacing the gRNA used to identify the location where the cut is made to a synthetic version called a bridged nucleic acid (BNA). The BNA is more stable and can guide Cas9 more carefully. First author of the study, PhD student Christopher Cromwell, said “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.”

The study – Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity – was recently published in the Journal Nature Communications.

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