Small and precise: these are the perfect traits for CRISPR systems, the Nobel Prize-winning technology used to alter nucleic acids such as RNA and DNA.
Rice University scientists have detailed the three-dimensional structure of one of the smallest known CRISPR-Cas13 systems used to shred or edit RNA, and their results have been utilized to further develop the tool to increase its precision. The molecule functions differently from other proteins in the same family, according to a study published in Nature Communications.
There are different types of CRISPR systems, and the one our research was focused on for this study is called CRISPR-Cas13bt3. The unique thing about it is that it is very small. Usually, these types of molecules contain roughly 1200 amino acids, while this one only has about 700, so that’s already an advantage.”
Yang Gao, Assistant Professor, Biosciences, Rice University
According to Gao, the small size is advantageous since it allows easier access and delivery to target-editing locations.
Contrary to Cas9 protein-associated CRISPR systems, which typically target DNA, Cas13 protein-associated systems target RNA, the intermediate “instruction manual” that converts the genetic data contained in DNA into a blueprint for building proteins.
Researchers intend to employ these RNA-targeting devices to combat viruses, which encode their genetic information using RNA rather than DNA.
Gao added, “My lab is a structural biology lab. What we are trying to understand is how this system works. So, part of our goal here was to be able to see it in three-dimensional space and create a model that would help us explain its mechanism.”
The scientists used a cryo-electron microscope to map the structure of the CRISPR system, laying the molecule on a thin coating of ice and sending an electron beam through it to gather data, which was then processed into a comprehensive three-dimensional model. They were taken aback by the results.
“We found this system deploys a mechanism that is different from that of other proteins in the Cas13 family. Other proteins in this family have two domains that are initially separated and, after the system is activated, they come together—kind of like the arms of a scissor—and perform a cut. This system is totally different: The scissor is already there, but it needs to hook onto the RNA strand at the right target site. To do this, it uses a binding element on these two unique loops that connect the different parts of the protein together,” Gao noted.
Xiangyu Deng, a postdoctoral research associate in the Yang Gao lab, stated that it was “really challenging to determine the structure of the protein and RNA complex. We had to do a lot of troubleshooting to make the protein and RNA complex more stable, so we could map it.”
After determining how the system works, researchers in the lab of chemical and biomolecular engineer Xue Sherry Gao intervened to fine-tune the system by assessing its activity and selectivity in real cells.
We found that in cell cultures these systems were able to hone in on a target much easier. What is really remarkable about this work is that the detailed structural biology insights enabled a rational determination of the engineering efforts needed to improve the tool’s specificity while still maintaining high on-target RNA editing activity.”
Xue Sherry Gao, Ted N. Law Assistant Professor, Chemical and Biomolecular Engineering, Rice University
Cellular experiments done by Emmanuel Osikpa, a research assistant in the Xue Gao lab, demonstrated that the modified Cas13bt3 targeted a specific RNA pattern with great fidelity.
I was able to show that this engineered Cas13bt3 performed better than the original system. Xiangyu’s comprehensive study of the structure highlights the advantage that a targeted, structurally guided approach has over large and costly random mutagenesis screening.”
Emmanuel Osikpa, Research Assistant, Rice University
Deng, X., et al. (2023). Structural basis for the activation of a compact CRISPR-Cas13 nuclease. Nature Communications. doi.org/10.1038/s41467-023-41501-5