CRISPR-Cas technology is widely utilized in scientific research and healthcare for the purposes of editing, inserting, deleting, or managing genes within living organisms. TnpB, a precursor to the famous “gene scissors,” is a much smaller protein, making it simpler to deliver into cells. Researchers have utilized protein engineering along with AI algorithms to improve TnpB’s functionality, enhancing its ability to edit DNA effectively. This development holds promise for addressing genetic disorders linked to high cholesterol in the future.
CRISPR-Cas technology sees extensive use in both research and medicine for editing, inserting, deleting, or controlling genetic material within organisms. TnpB, a predecessor of the famed “gene scissors,” boasts a smaller size, which permits easier cellular transport. Recent advancements by researchers at UZH, leveraging protein engineering and artificial intelligence, have significantly boosted TnpB’s efficiency in DNA editing, potentially offering solutions for genetic high cholesterol issues in the future.
The CRISPR-Cas systems, comprising protein and RNA elements, initially functioned as a natural defense mechanism in bacteria against invading viruses. Over the past ten years, the re-engineering of these “gene scissors” has transformed genetic engineering practices in both scientific and medical fields. These tools can be programmed to locate specific DNA regions and edit genetic information accurately, enabling the correction of harmful mutations in the DNA, restoring it to a healthy form.
A More Compact Genome Editing Tool
Recent findings indicate that Cas proteins originated from much smaller proteins, with TnpB being a direct ancestor of Cas12. The bulkiness of Cas proteins presents challenges in delivering them to the correct cells within the body, prompting recent investigations into using their smaller evolutionary relatives for genome editing. However, the smaller alternatives often have less efficiency. This challenge has now been successfully addressed by a research team led by Gerald Schwank at the Institute of Pharmacology and Toxicology at the University of Zurich (UZH), in collaboration with experts from ETH Zurich. “Through engineering the diminutive yet potent TnpB protein, we developed a variant that demonstrates a 4.4-fold boost in DNA modification efficiency, enhancing its efficacy as a gene editing tool,” states Schwank.
TnpB proteins are found in a variety of bacterial and archaeal species, with the studied TnpB originating from the resilient bacterium Deinococcus radiodurans. This organism can withstand extreme environmental conditions like cold, desiccation, vacuum, and acidic environments, making it one of the most radiation-resistant known life forms. Previous studies have indicated that the compact TnpB protein could be utilized for genome editing in human cells, although its initial efficacy and targeting capabilities were limited due to strict requirements for DNA recognition.
Improved Binding Capability and Expanded DNA Target Sequences
To optimize TnpB for better DNA editing in mammalian cells, researchers enhanced its functionality compared to the standard variant. “The key was to modify the protein in two significant ways: first, to increase its efficiency in reaching the nucleus where the genomic DNA resides, and second, to allow it to target a wider array of genome sequences,” explains Kim Marquart, a PhD student in Schwank’s lab and the primary author of the research.
In order to ascertain the features in DNA sequences that affect genome editing efficiency, the researchers evaluated TnpB across 10,211 distinct target sites. Collaborating with Michael Krauthammer’s team at UZH, they engineered a novel artificial intelligence model capable of predicting TnpB’s editing effectiveness at various target locations. “Our model can anticipate TnpB’s performance under different conditions, facilitating the design of successful gene editing experiments. With these predictions, we achieved up to 75.3% efficiency in mouse livers and 65.9% in mouse brains,” adds Marquart.
Gene Editing Therapy for Cholesterol Genetic Defect
“We were able to use effectively transportable Adeno-associated viral vectors to deliver the tools into mouse cells during the animal experiments. Thanks to its compact size, the TnpB gene editing system can fit into a single virus particle,” states Marquart. Conversely, multiple virus particles are required for the CRISPR-Cas9 components, leading to the necessity for higher doses of vectors.
In their ongoing research, the team investigated the application of the TnpB tool to treat familial hypercholesterolemia, a genetic disorder that results in persistently elevated cholesterol levels and affects around 31 million individuals worldwide. This condition raises the risk of early atherosclerotic cardiovascular diseases. “We successfully edited a gene involved in cholesterol regulation, resulting in nearly an 80% reduction in cholesterol levels in treated mice. Our goal is to translate similar gene editing techniques for human patients suffering from hypercholesterolemia,” concludes Schwank.
