Sleek CRISPR systems get almost all the attention. They rely on single-protein nucleases instead of multiunit effectors, which are, presumably, too unwieldy for gene engineering applications. Yet CRISPR jumbles have been given a tumble by scientists at Duke University. Led by Charles Gersbach, PhD, the Rooney Family associate professor of biomedical engineering and Adrian Oliver, PhD, a postdoctoral fellow, these scientists used a multiunit effector system to turn target genes on and off in human cells.
Specifically, the scientists used a class 1 CRISPR-Cas system called Cascade (CRISPR-associated complex for antiviral defense). And as if it wasn’t clunky enough already, the scientists tacked on a couple of extras—activation and repression domains. The system, however, omitted the Cas enzyme that would have ordinarily been present.
In this case, the Cas enzyme would have been Cas3, a sort of molecular shredder. Leaving it out seemed a good idea, since the scientists were experimenting with gene regulation, something rather more delicate than shredding.
Using modified versions of Cascade systems from Escherichia coli and Listeria monocytogenes, the scientists achieved both DNA targeting and transcriptional control. They presented their findings in a paper (“Targeted transcriptional modulation with type I CRISPR–Cas systems in human cells”) that appeared September 23 in Nature Biotechnology.
“We validate Cascade expression, complex formation, and nuclear localization in human cells, and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome,” the article’s authors wrote. “By tethering activation and repression domains to Cascade, we modulate the expression of targeted endogenous genes in human cells.”
Class 1 CRISPR systems, which include the type I CRISPR-Cas system in the current study, represent about 90% of all CRISPR systems in nature. Yet these systems remain largely unexplored for genome engineering applications. By demonstrating the potential of repurposed type I CRISPR-Cas systems, the Duke scientists hope to open a new and diverse frontier of genome engineering technology.
“We have found Cascade’s structure to be remarkably modular, allowing for a variety of sites to attach activators or repressors, which are great tools for altering gene expression in human cells,” said Oliver, the lead author of the study. “The flexible nature of Cascade makes it a promising genome engineering technology.”
“[Our purpose] was to explore the diversity of CRISPR systems,” added Gersbach, the study’s senior author. “There have been thousands of papers about CRISPR-Cas9 in the last decade, and yet we’re constantly learning new things about it. With this study, we’re applying that mindset to the other 90% of what’s out there.”
So far, the Duke team has shown that Class 1 systems are comparable to CRISPR-Cas9 in terms of accuracy and application. Going forward, the team intends to explore how these systems differ from their Class 2 counterparts, and how these differences could prove useful for biotechnology applications.
The team is also interested in studying how Class 1 systems could address general challenges for CRISPR-Cas research, especially issues that complicate potential therapeutic applications, like immune responses to Cas proteins and concurrently using multiple types of CRISPR for different genome engineering functions.
“We know CRISPR could have a big impact on human health,” noted Gersbach. “But we’re still at the very beginning of understanding how CRISPR is going to be used, what it can do, and what systems are available to us. We expect that this new tool will enable new areas of genome engineering.”