CRISPR systems have revolutionized nucleic acid editing, and Rice University scientists have made a significant stride in this field by detailing the 3D structure of one of the smallest CRISPR-Cas13 systems known. In their study published in Nature Communications, the researchers not only examined this diminutive system but also improved its precision. This particular molecule, CRISPR-Cas13bt3, sets itself apart from other proteins in the CRISPR family.
Lead researcher Yang Gao, an assistant professor of biosciences and Cancer Prevention and Research Institute of Texas Scholar, highlighted the uniqueness of CRISPR-Cas13bt3, emphasizing its compact size. While typical CRISPR molecules contain around 1200 amino acids, this system boasts only about 700, providing an advantage due to better access and delivery to target-editing sites.
CRISPR-Cas13-associated systems, in contrast to Cas9-associated systems that target DNA, home in on RNA—the intermediary “instruction manual” that translates genetic information into protein assembly instructions. Scientists anticipate that RNA-targeting systems like these could be potent tools in combating viruses, which often use RNA for genetic encoding.
Yang Gao’s structural biology lab played a pivotal role in comprehending the system’s workings. By employing a cryo-electron microscope, they meticulously mapped the structure of CRISPR-Cas13, creating a detailed 3D model. The results yielded an unexpected revelation: this system operates differently from its Cas13 protein family counterparts.
In other Cas13 proteins, two initially separated domains come together like scissor blades when the system is activated, leading to a cut. CRISPR-Cas13bt3, however, employs a distinct mechanism. The “scissor” is already present, but it requires attachment to the RNA strand at the precise target site. To achieve this, it utilizes binding elements on two unique loops connecting different parts of the protein.
Xiangyu Deng, a postdoctoral research associate, highlighted the challenges in determining the complex protein and RNA structure, requiring significant troubleshooting to stabilize it for mapping.
Once the structural insights were gained, researchers from chemical and biomolecular engineer Xue Sherry Gao’s lab worked on enhancing the system’s precision. Their efforts involved testing the system’s activity and specificity in living cells.
The outcomes were promising, as the systems showcased a greater ability to target specific sites within cell cultures. Sherry Gao, the Ted N. Law Assistant Professor of Chemical and Biomolecular Engineering, emphasized that the work’s remarkable aspect lies in the structural biology insights guiding rational engineering improvements while maintaining high RNA editing activity.
Emmanuel Osikpa, a research assistant in the Xue Gao lab, confirmed the enhanced performance of the engineered Cas13bt3 in cellular assays, demonstrating its superior fidelity in targeting designated RNA motifs compared to the original system.
In summary, the Rice University researchers have delved into the intricacies of a compact CRISPR-Cas13 system, shedding light on its unique mechanisms and harnessing this knowledge to enhance precision in nucleic acid editing.