Researchers at the University of California have discovered four CRISPR-Cas9 inhibitors, which could potentially be used to switch off the gene-editing system to prevent unintentional gene edits.
The CRISPR-Cas9 system has attracted considerable interest from scientists over the past decade. The system is used by bacteria as a protection against viral infections. Since the system was identified, it has shown tremendous potential for therapeutic use, allowing highly targeted changes to be made to the genomes of living cells. While other methods of genome editing exist, the beauty of CRISPR-Cas9 is its simplicity, versatility, and precision. The system has been shown to allow single point mutations to be triggered in a target gene via a single gRNA.
The gRNA is used to bind to a very specific DNA sequence, then the Cas9 enzyme is used to cut the DNA strands in a precise location. The DNA damage is recognized, and cells then repair their DNA; however, it is possible to introduce desirable changes using cells’ DNA repair machinery.
However, while the system has been used in humans and has been shown to be incredibly precise, it is not without its disadvantages. Desirable changes can be made, although there is potential for unintended cuts to be made to DNA. Those off-target cuts could potentially cause patients harm.
There has been considerable focus in the past few years on improving the precision of CRISPR-Cas9, such as identifying a mechanism for turning off the CRISPR-Cas9 system. If scientists are able to turn CRISPR-Cas9 off, it could be used as a failsafe to prevent unwanted cuts.
Now, Dr. Joseph Bony-Denomy and his team at the University of California’s Department of Microbiology and Immunology and the Quantitative Biosciences Institute have identified CRISPR-Cas9 inhibitors that could be used as an off switch for the system.
Bony-Denomy and his team were working on Listeria bacteria with inactive CRISPR-Cas9 systems. The team were looking for bacterial strains that had viral genes inserted into their DNA. If viruses were capable of bypassing the bacterial CRISPR-Cas9 system, it was hypothesized that those bacteria must have a mechanism in place to turn off the Cas9 protein, otherwise the viral DNA would have been removed and the DNA repaired.
More than 300 bacterial strains were examined and approximately 3% were found to have some type of CRISPR-Cas9 inhibitors. Four such proteins have now been identified that have been shown to inhibit CRISPR-Cas9 activity, two of which – AcrIIA2 and AcrIIA4 – have been shown to inhibit the SpyCas9 (Streptococcus pyogenes) protein used by many labs to make DNA cuts.
The next step is for the team to show how these CRISPR-Cas9 inhibitors can be used to control the CRIPSR mechanism and improve the precision of gene editing in human cells. The team are also working on identifying the mechanism by which these proteins inhibit Cas9 activity, as well as continuing to search for better CRISPR-Cas9 inhibitors in other bacteria.