CRISPR-Cas systems in bacteria utilize RNA molecules and CRISPR-associated (Cas) proteins to target and destroy the DNA of invading viruses. This discovery has led to a revolution in gene-editing technology. This class 2 system is the most widely utilized genome editing tool today because of their simplistic design, relying only on one Cas protein for DNA cleavage. However, in the bacterial world, it is much less uncommon than other CRISPR classes.

The goal of the current research was to harness class 1 CRISPR systems to turn target genes on and off and edit the epigenome in human cells for the first time. The efforts were led by Charles Gersbach, the Rooney Family Associate Professor of Biomedical Engineering at Duke, and Adrian Oliver, a post-doctoral fellow in the Gersbach lab who led the project.

Class 1 CRISPR systems are not so simple, because they rely on multiple proteins working together in a complex called Cascade (CRISPR-associated complex for antiviral defense) to target DNA. After binding, Cascade recruits a Cas3 protein that cuts the DNA. "The flexible nature of Cascade makes it a promising genome engineering technology," said Oliver.

The team provided evidence that these class 1 systems are comparable to CRISPR-Cas9 in terms of accuracy and application. As they consider future directions, they are curious to explore how these systems differ from their class 2 counterparts, and how these differences could prove useful for biotechnology applications.

To test the effectiveness of the system, the researchers attached gene activators to specific sites along a type I E. coli Cascade complex and targeted the system to bind gene promoters, which regulate gene expression levels. This showed that the Cascade activator not only binds to the correct site and can turn up the levels of the target gene but does so with accuracy and specificity comparable to CRISPR/Cas9. Alternatively, the team also showed that the activator domain could be swapped for a repressor to turn target genes off. Again, the researchers noted accuracy and specificity comparable to CRISPR/Cas9 methods.

This system was tested in type I variants of class 1 CRISPR–Cas systems from Escherichia coli and Listeria monocytogenes, which target DNA via a multi-component RNA-guided complex termed Cascade. The engineers also proved that class 1 CRISPR–Cas systems can be expressed in mammalian cells and used for DNA targeting and transcriptional control.

"If you were to look at the individual CRISPR systems of all the bacteria in the world, nearly 90 percent are class 1 systems," said Gersbach. "CRISPR-Cas biology is an incredible source for biotechnology tools, but until recently everyone has only been looking at a small slice of the pie." Now, the team is optimistic that their study, and the related work of others in the field, will promote other researchers to investigate class 1 CRISPR systems.

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