Reducing the side-effects of CRISPR gene editing

The revolutionary CRISPR gene-editing technology holds huge potential to cure all manner of genetic diseases and disorders, efficiently deleting and replacing faulty genes at a fraction of the cost of other gene therapies. But the bright shining path to a brave new world is bumpier in reality than theory, with the precision editing technology proving somewhat less precise in real life than in the petri dish.

Research published earlier this year pointed to the potential of the technology to introduce hundreds of unintended mutations into the genetic material of treated organisms. This was discovered by sequencing the whole genome of mice that had previously undergone CRISPR gene editing.

A fix to control the problem of unintended gene editing, however, may come sooner rather than later, with scientists predominantly from the University of California Berkeley reporting a potential way to reduce off-target cutting during the genome editing process.

Led by Jiyung Shin, of UCB’s Innovative Genomics Institute, the team report identification of an inhibitor protein that can block the function of the “molecular scissors” that enable CRISPR technology to cut strands of DNA in order to remove and replace parts for the genome. Those scissors are an enzyme known as Cas9 – short for “CRISPR associated protein 9” and the reason the gene-editing technology is commonly referred to as CRISPR-Cas9.

The inhibitor protein comes from a listeria bacteriophage. This new research suggests the protein known as AcrIIA4 could, along with other natural inhibitor proteins, help refine gene editing attempts based on CRISPR and Cas enzymes.

“The recent and rapid expansion of the Cas9 toolkit for gene-editing applications has lacked an inducible off switch to prevent undesired gene editing,” the researchers state in their paper, published in Science Advances.  “Newly discovered protein inhibitors, encoded by bacteriophages, provide an attractive solution to this problem because these proteins are small and function well in human cells.”

Their research, they report, demonstrates that AcrIIA4, “the most potent SpyCas9 inhibitor in human cells”, acts as a DNA mimic, sitting on the target DNA at exactly the right point to block the Cas9 enzyme from recognising it as the place to it wants to attach and cut.

Simply blocking CRISPR from working at all, however, wouldn’t be much help, so what makes this new research potentially very useful is that the researchers outline a way to time the use of the phage so its blocking effect isn’t total. While the presence of the inhibitor prior to any CRISPR operation “almost completely abolishes overall gene editing”, its “timed addition” after initiating gene editing can influence the amount of time that Cas9 is active in the nucleus, “thereby selectively limiting off-target editing”.

Thus this “anti-CRISPR DNA mimic” could be the saviour of CRISPR as a remedial therapy, with the inhibitor “broadly useful in situations where precise control of either on- or off-target gene editing is desirable”. That would be, one suspects, just about every situation.