New technique makes large-scale gene editing possible in animals, shortening labor time by years

Working on tiny worms, scientists can now test the effects of thousands of genetic mutations at once.

A new gene-editing technique developed by researchers at the University of Oregon (UO) compresses what previously would have been years of work into just days, making new kinds of research possible in animal models. This will allow biologists to do experiments that compare many versions of a gene, looking for mutations that lead to specific traits and tracking how they change over time.

This research is often a first step towards identifying mutations relevant to human health or to unraveling the mechanisms underlying human disease.

While mass gene-editing tricks have been developed for single-celled organisms like bacteria and yeast, this is the first time it’s been possible on this scale in an animal.

“In biology, we spend a lot of time working with genetic mutants. But in animals, we’re limited by how many genetic mutants we can create at once,” says Zach Stevenson, a graduate student in Patrick Phillips’ lab. at the OU. who helped design the technique. “It’s a way around that bottleneck.”

Stevenson and colleagues describe their new technique in a preprint published on bioRxiv.

They piloted the system in C. elegans, a small worm that is a popular species for biological research. A similar approach could possibly work in other laboratory animals, such as flies or mice, Stevenson said.

“Genetic engineering of the DNA of microbes has served as the basis for the revolution in biotechnology over the past three decades, but it has been difficult to do so at scale in animal systems,” Phillips said. “The new approach developed in our lab can serve as the platform for a whole new way to use a simple animal as the basis for synthetic biology in the same way that bacteria and yeast have been used for a generation.”

There are many reasons why scientists might want the ability to create multiple genetic mutations at once. For example, they might be looking for a mutation that might make an animal resistant to a specific drug, or better able to survive under certain conditions, or less susceptible to a disease. They might need to screen dozens or even hundreds of possible variations on a gene to find the most effective one.

These types of experiments are extremely slow in animals. Each mutant strain – a set of worms with a specific genetic modification – must be modified individually. Making a mutant “usually takes between seven and 10 hours of hands-on work,” Stevenson said. Using this new system, “for the same job of doing three or four mutations, you can do tens of thousands”.

To speed things up, Stevenson and his colleagues devised a way to compress hundreds or even thousands of possible mutations into a single “library”. Each book in the library is a small snippet of genetic code, meaningless and non-functional on its own. Each extract fits into an artificial gap in the targeted gene, like a Mad Libs genetic puzzle.

This design means that instead of individually injecting many individual worms with different versions of a gene, researchers can inject the entire library of mutations into a single worm.

Then, when the worm reproduces, the library grows. In each offspring, a book from the mutation library is randomly selected to complement the targeted gene. When a segment of the gene library slips through, it makes the gene active, like flipping a switch to complete an electrical circuit.

The result: a collection of worms that all have different randomly selected genetic mutations.

The researchers named their technique TARDIS, a playful nod to Dr Who’s time and space traveling police box. Here it stands for Transgenic Arrays Resulting in Diversity of Integrated Sequences. Like the fictional TARDIS, the worm “is bigger on the inside,” says Stevenson. (That is, it contains a lot of extra genetic material.)

The researchers tested TARDIS with a gene that gives worms resistance to antibiotics. But they see broad applications for biology in general, including research on other model organisms.

This could be particularly useful for studying interactions between proteins or signaling between cells, suggests UO research professor Stephen Banse, who helped develop TARDIS. Such interactions are often relevant to understanding disease, but scientists lose important context by studying them in yeast or bacteria, Banse said. “Now we can do these things in an animal model.”