Exploring the enhanced efficiency of CRISPR/Cas9 gene editing with interstrand crosslinks

Wednesday - May 17 2023, 00:15 UTC - 1 year ago

By introducing interstrand crosslinks into the CRISPR/Cas9 workflow, researchers in the University of California, Santa Barbara have managed to threefold increase the efficiency of gene editing without increasing mutation frequencies or altering end-joining repair outcomes.

Gene editing is a powerful method for both research and therapy. Since the advent of the Nobel Prize-winning CRISPR/Cas9 technology, a quick and accurate tool for genome editing discovered in 2012, scientists have been working to explore its capabilities and boost its performance.

Researchers in the University of California, Santa Barbara biologist Chris Richardson’s lab have added to that growing toolbox, with a method that increases the efficiency of CRISPR/Cas9 editing without the use of viral material to deliver the genetic template used to edit the target genetic sequence. According to their new paper published in the journal Nature Biotechnology, their method stimulates homology-directed repair (a step in the gene editing process) by approximately threefold "without increasing mutation frequencies or altering end-joining repair outcomes." .

"We’ve found a chemical modification that improves non-viral gene editing and also discovered an intriguing new type of DNA repair," Richardson said.

The CRISPR/Cas9 method works by capitalizing on a defense technique employed by bacteria against viral attackers. To do this, the bacteria snip a piece of the invading virus’s genetic material, and incorporate it into their own in order to recognize it later. Should the bacteria get reinfected, they can target the now-familiar genetic sequences for destruction.

In gene editing, this process uses the enzyme Cas9 as molecular "scissors" to snip sequences it recognizes, guided by the CRISPR system. This cut is also an opportunity to replace the severed genes with similar (homologous) but improved ones, utilizing the cell’s natural repair mechanisms. If successful, the cell should have modified expressions and functions thereafter.

To deliver the repair template DNA to the nucleus of the cell where its genetic material lives, oftentimes viruses are used. While they are effective, the researchers say, viral workflows "are expensive, difficult to scale and potentially toxic to cells." .

Nonviral templates are potentially less expensive and more scalable, although researchers still must overcome efficiency and toxicity barriers. In their study, the Richardson Lab found that introducing interstrand crosslinks into the workflow increased homology directed repair dramatically.

"Every workflow that we have put this approach into has worked better by roughly threefold," Richardson said.

Interstrand crosslinks are lesions that keep the double strands of a DNA helix tethered to each other, making them unable to replicate. Cancer chemotherapies use this mechanism to interrupt tumor growth and kill cancer cells. Added to a homology directed repair template, however, these crosslinks were found to stimulate the cell’s natural repair mechanisms and increase the likelihood of editing success.

"Basically, what we’ve done is taken this template DNA and damaged it," Richardson said. "We’ve in fact damaged it in the most severe way I can think of. And the cell does something that is quite surprising. It repairs it almost perfectly. And that’s all CRISPR needs to work." .