Researchers are finding ways to improve nonviral gene editing as well as new types of DNA repair

(Nanowerk News) Gene editing is a powerful method for research and therapy. Since the emergence of the Nobel Prize-winning CRISPR/Cas9 technology, a fast and accurate tool for genome editing discovered in 2012, scientists have been working to explore its capabilities and improve its performance.

Researchers in the lab of UC Santa Barbara biologist Chris Richardson have added to that growing toolbox a method that increases the efficiency of CRISPR/Cas9 editing without using viral material to deliver the genetic template used to edit the target genetic sequence. According to their new paper published in the journal Natural Biotechnology (“Interstrand cross-linking of homologous repair DNA templates enhances gene editing in human cells”), their method stimulated homology-directed repair (a step in the gene editing process) approximately threefold “without increasing the mutation frequency or altering the final splice repair outcome”.

Find, Cut and Paste

The CRISPR/Cas9 method works by utilizing the defense techniques used by bacteria against viral invaders. To do this, the bacteria take a piece of the invading virus’s genetic material, and incorporate it into their own genetic material in order to recognize it later. If bacteria are reinfected, they can target destruction of the now-familiar genetic sequence.

In gene editing, this process uses the Cas9 enzyme as a molecular “scissor” to cut the sequence it recognizes, guided by the CRISPR system. This truncation is also an opportunity to replace the truncated gene with a similar (homologous) but better gene, leveraging the cell’s natural repair mechanisms. If successful, the cell should modify the expression and function afterwards.

To deliver the repair template DNA to the cell nucleus where its genetic material lives, viruses are often used. While effective, say the researchers, viral workflows are “expensive, difficult to quantify, and potentially toxic to cells.”

Nonviral templates are potentially cheaper and more scalable, although researchers still have to overcome efficiency and toxicity hurdles. In their study, the Richardson Lab found that incorporating interstrand crosslinks into workflows dramatically increased homology directional improvements.

“Every workflow we’ve done with this approach has worked roughly three times as well,” says Richardson.

Interstrand cross-links are lesions that make the double strands of the DNA helix anchored to one another, rendering them unable to replicate. Cancer chemotherapy uses this mechanism to stop tumor growth and kill cancer cells. Added to the homology-directed repair template, however, this cross-linking was found to stimulate the cell’s natural repair mechanisms and increase the likelihood of successful editing.

“Basically, what we do is take this template DNA and damage it,” says Richardson. “We have actually damaged it in the most severe way I can think of. And the cell doesn’t say, ‘Hey, this is trash; let me throw it away.’ What the cell is really saying is, ‘Hey, this looks good; let me put it in my genome.’” The result is a highly efficient, error-prone, nonviral gene-editing system.

Their discovery, like many breakthroughs in science, was actually a happy coincidence. While working to purify proteins to study DNA repair, graduate student researcher and lead author Hannah Ghasemi noted an unexpected change in their experimental results.

“We introduced these chemical modifications to the DNA template in order to be able to pull it out of the cell and see what proteins were bound to it, and I just checked to see if these modifications affect the editing in any capacity. ,” he said. “I was hoping to see no changes or it might actually have had a negative impact on the edits.”

What he found was a positive effect, up to three times the editing activity of the uncrosslinked control. Additionally, the team found that even with an increase in edits—and therefore the likelihood of errors—there was no increase in mutation frequency. They’re still investigating the specific mechanisms that lead to these results, but they have an idea.

“What we think is happening is the cell detects and tries to repair the damaged DNA that we’ve added with these crosslinks,” said Richardson. “And in doing so, it delays the cell from passing through checkpoints where it would normally stop this recombination process. So by extending the amount of time it takes for cells to do this recombination, the more likely it is that edits will complete. Learning this new process could also lead to a better understanding of how cells detect editing reagents and how they “decide” to accept them or not, he said.

This method will be most widely used in ex-vivo gene editing applications, according to the team, namely in the realm of disease research and preclinical work.

“We can more effectively knock down a gene and insert something into the genome to study systems outside the human body in a laboratory setting,” said Ghasemi. This development allows them to more efficiently build disease models and test hypotheses about how disease works, which can lead to better clinical and therapeutic approaches.