New Gene Editing Tool Shows Promise for Treating Diseases

Bacterial and archaeal CRISPR-Cas systems, comprising protein and RNA components, were initially developed as a natural defense mechanism against invading viruses. Over the past decade, the reconfiguration of these so-called “genetic scissors” has revolutionized the field of genetic engineering in both scientific research and medical applications.

These tools can be programmed to target a specific location within the DNA and precisely modify the genetic information. For instance, a disease-causing mutation in the DNA can be reverted to its healthy counterpart.

Much Smaller Genome Editing Tool

Researchers have uncovered that Cas proteins, the key components of genome editing tools, have evolved from much smaller precursor proteins, with TnpB being the ancestral form of Cas12.

The large size of Cas proteins poses challenges in delivering them to target cells, prompting scientists to explore the use of their smaller evolutionary predecessors as alternative genome editing tools. However, these smaller variants have been found to function less efficiently.

A research team led by Gerald Schwank from the Institute of Pharmacology and Toxicology at the University of Zurich (UZH), in collaboration with colleagues from the ETH Zurich, has now addressed this challenge.

By engineering the small but powerful protein TnpB, we were able to design a variant that shows a 4.4-fold increase in efficiency of modifying DNA – making it more effective as a gene editing tool.”

Gerald Schwank, Institute of Pharmacology and Toxicology, University of Zurich

The TnpB proteins are widely distributed across various bacterial and archaeal species. The specific TnpB studied by the researchers was derived from the bacterium Deinococcus radiodurans.

This microorganism is renowned for its exceptional resilience, withstanding extreme conditions such as cold, dehydration, vacuum, and acidity. It is also one of the most radiation-tolerant organisms known to humanity.

The compact TnpB protein has been previously demonstrated to function in genome editing within human cells, although its efficiency and targeting capabilities have been limited due to the specific requirements for DNA binding.

Better Binding Ability and Broader Range of DNA Target Sequences

The researchers have optimized the TnpB protein to enhance DNA editing capabilities in mammalian cells. The modified version of TnpB demonstrates improved efficiency compared to the original protein when performing DNA editing tasks in mammalian cellular environments.

The trick was to modify the tool in two ways: first, so that it more efficiently goes to the nucleus where the genomic DNA is located, and second, so that it also targets alternative genome sequences.”

Kim Marquart, PhD Student and Study First Author, University of Zurich

To determine the specific DNA sequence features that influence the efficiency of genome editing using the TnpB system, the researchers evaluated its performance across 10,211 different target sites. Collaborating with the team led by Professor Michael Krauthammer from the University of Zurich, the researchers developed a novel artificial intelligence model that can accurately predict the editing efficiency of TnpB at any given target site.

 “Our model can predict how well TnpB will work in different scenarios, making it easier and faster to design successful gene editing experiments. Using these predictions, we achieved up to 75.3% efficiency in mouse livers and 65.9% in mouse brains,” Marquart added.

Gene Editing Therapy of Genetic Defects for High Cholesterol

“For the animal experiments, we were able to use clinically viable Adeno-associated viral vectors to efficiently transport the tools into mouse cells. Due to its small size, the TnpB gene editing system can be packaged into a single virus particle,” Marquart said.

In the case of CRISPR-Cas9, the necessary components must be encapsulated into multiple virus particles, necessitating the administration of higher doses of the vector.

In the present study, the researchers investigated the potential of the TnpB tool for the treatment of familial hypercholesterolemia. This genetic disorder is characterized by persistently elevated cholesterol levels and affects approximately 31 million individuals worldwide. The condition is associated with an increased risk of early-onset atherosclerotic cardiovascular disease.

We were able to edit a gene that regulates cholesterol levels, thereby reducing the cholesterol in treated mice by nearly 80%. The goal is to develop similar gene editing strategies in humans to treat patients suffering from hypercholesterolemia.”

Gerald Schwank, Institute of Pharmacology and Toxicology, University of Zurich