Researchers at the Salk Institute have described for the first time the molecular structure of CRISPR-Cas13d, an enzyme for emerging RNA-editing technology. They were able to visualize the enzyme with cryo-electron microscopy (cryo-EM) as reported in an article (“Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d”) in Cell.
“CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively,” write the investigators.
“Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.”
"This paper provides a molecular blueprint for RNA-targeted genetic engineering," says Salk assistant professor Dmitry Lyumkis, Ph.D., a structural biologist and one of the corresponding authors of the study. "It adds to the breadth of tools that are needed for conducting this kind of crucial biomedical research."
In the CRISPR-Cas9 system, Cas9 cuts DNA. Having editing tools for RNA, however, would allow scientists to modify a gene's activity without making a permanent—and potentially dangerous—change to the gene itself, according to the researchers.
"DNA is constant, but what's always changing are the RNA messages that are copied from the DNA," says Salk research associate Silvana Konermann, Ph.D., a Howard Hughes Medical Institute Hanna Gray Fellow and one of the study's first authors. "Being able to modulate those messages by directly controlling the RNA has important implications for influencing a cell's fate."
Earlier this year, Dr. Konermann and Helmsley-Salk Fellow Patrick Hsu, Ph.D., published another paper in Cell discovering the family of enzymes called CRISPR-Cas13d and reporting that this alternate CRISPR system was effective in recognizing and cutting RNA. The team also showed this tool could be used to correct a disease-causing protein imbalance in cells from a person with dementia.
The new study, a collaboration between the Lyumkis and Hsu labs, built on the discovery of the Cas13d family and provides the molecular details that explain how it works.
"In our previous paper, we discovered a new CRISPR family that can be used to engineer RNA directly inside of human cells," says Dr.Hsu, who is the other corresponding author of the new work. "Now that we've been able to visualize the structure of Cas13d, we can see in more detail how the enzyme is guided to the RNA and how it is able to cut the RNA. These insights are allowing us to improve the system and make the process more effective, paving the way for new strategies to treat RNA-based diseases."
The team used cryo-EM to reveal new details into Cas13d by freezing the enzyme in different, dynamic states, allowing researchers to decode a range of activities instead of just seeing one activity at a single point in time.
"This enabled us to see how Cas13d guides, binds and targets the RNA," says Cheng Zhang, Ph.D., a research associate in the Lyumkis lab and the paper's other first author. "We hope this new knowledge will help expand on the power of gene-editing tools."