A new discovery by UC San Francisco researchers has the potential of greatly improving the widely used CRISPR-Cas9 gene-editing system.
According to a study published in Cell, researchers have effectively discovered an off-switch for the CRISPR-Cas9 gene-editing system. The off-switch are the newly identified anti-CRISPR proteins that are produced by bacterial viruses. Originally evolved in bacteria as an immune system to protect against viral infections, CRISPR-Cas9 is being increasingly used an a gene editing system, enabling scientists to quickly and efficiently modify genetic information and tweak gene activity in virtually any organism.
Multiple applications have been envisaged for this gene-editing system, but one of the major issues with the technology is precision and scientists haven’t been able to ensure that there are no unintended changes to the genetic material. However, the newly discovered anti-CRISPR proteins could hold the answer to precision.
According to researchers the proteins are the first to work against the type of CRISPR-Cas9 system most commonly used by laboratories and the gene editing industry and could help resolve precision problems as well as provide a fail-safe to quickly block any potentially harmful uses of the technology.
To find the anti-CRISPR protein researchers came up with a clever trick: They reasoned that they should be able to identify bacteria with inactivated CRISPR systems by looking for evidence of so-called “self-targeting” — bacterial strains where some virus had successfully gotten through the Cas9 blockade and inserted its genes into the bacterial genome. The team hypothesized that these phages must encode some anti-CRISPR agent, or else Cas9 would kill the bacteria by cutting its own genome where the viral DNA had been inserted.
Using a bioinformatics approach the team examined nearly 300 strains of Listeria, a bacterial genus famous for its role in food-borne illness, and found that three percent of strains exhibited “self-targeting”. Further investigation isolated four distinct anti-CRISPR proteins that proved capable of blocking the activity of the Listeria Cas9 protein, which is very similar to SpyCas9.
Additional experiments showed that two of the four anti-CRISPR proteins — which the researchers dubbed AcrIIA2 and AcrIIA4 — worked to inhibit the ability of the commonly used SpyCas9 to target specific genes in other bacteria – such as E. coli – as well as in engineered human cells. Together, the results suggest that AcrIIA proteins are potent inhibitors of the CRISPR-Cas9 gene editing system as it has been adopted in labs around the world.
Researcher believe that the ability to deactivate SpyCas9 will make CRISPR-based gene editing much safer and more precise by resolving the ongoing problem of unintended “off-target” gene modifications, which become more likely the longer the CRISPR gene editing machinery remains active in target cells.