Scientists have studied in detail the three-dimensional structure of one of the smallest known CRISPR-Cas13 systems, CRISPR-Cas13bt3, which is used for RNA modification and operates differently from other proteins in the same family. This discovery allows them to improve the precision of the tool, allowing for better access and delivery to the target editing site, potentially allowing them to fight viruses more effectively by targeting RNA.
Rice University scientists have detailed the three-dimensional structure of one of the smallest known CRISPR-Cas13 systems for chopping or modifying RNA, and used their findings to further modify the tool to improve its precision. According to a study published in Nature Communications, the molecule works differently than other proteins in the same family.
"There are different types of CRISPR systems, and the focus of our study this time is one called CRISPR-Cas13bt3," said Yang Gao, assistant professor of biological sciences and a scholar at the Cancer Prevention and Research Institute of Texas who helped lead the study. "What's unique about it is that it's very small. Typically, molecules like this contain about 1,200 amino acids, and this one only has about 700, so that's already an advantage."
The small size is an advantage as it allows for better access and delivery to the target editing site. Unlike CRISPR systems associated with the Cas9 protein, which typically targets DNA, Cas13-related systems target RNA, which is the intermediate "instructions" for converting the genetic information encoded in DNA into a blueprint for assembling a protein.
The researchers hope these RNA-targeting systems could be used to fight viruses, which typically use RNA rather than DNA to encode their genetic information.
"My laboratory is a structural biology laboratory," Gao Yang said. "We're trying to understand how this system works. So part of our goal is to be able to see it in three dimensions and create a model that helps us explain its mechanisms."
The researchers used cryo-electron microscopy to map the structure of the CRISPR system, placing the molecule on a thin layer of ice and firing a beam of electrons through it, generating data that was then processed into a detailed three-dimensional model. The results surprised them.
"We found that this system deploys a different mechanism than other proteins in the Cas13 family, which have two domains that initially separate, and after the system is activated, they come together - a bit like the two arms of scissors - and make the cut. This system is completely different: the scissors already exist, but it needs to hook the RNA strand at the right target site. To do this, it uses a binding element on these two unique loops to connect different parts of the protein together."
Xiangyu Deng, a postdoctoral associate in Gao Yang's lab, said, "Determining the structure of the protein and RNA complex is really challenging, and we have to do a lot of troubleshooting to make the protein and RNA complex more stable so that we can map its structure."
Once the team figured out how the system worked, researchers in the laboratory of chemical and biomolecular engineer Shirley Gao began tweaking the system to improve its accuracy by testing its activity and specificity in living cells.
"We found that these systems were able to target more easily during cell culture," said Sherry Gao, Ted N. Law Assistant Professor of Chemical and Biomolecular Engineering. "What's really valuable about this work is that the detailed structural biology insights allowed us to rationally identify the engineering efforts required to improve the specificity of the tool while still maintaining high target RNA editing activity."
Emmanuel Osikpa, a research assistant in Xue Gao's laboratory, conducted cell experiments and confirmed that engineered Cas13bt3 can target specified RNA groups with high fidelity.
"I was able to show that this engineered Cas13bt3 performed better than the original system," Osikpa said. "Comprehensive study of the structure highlighted the advantages of a targeted, structure-driven approach over large-scale, costly random mutagenesis screens."