Gene editing is a powerful technique widely used in research and therapy. The groundbreaking CRISPR/Cas9 technology, which won the Nobel Prize in 2020, has revolutionized genome editing since its discovery in 2012. Scientists have been striving to explore its capabilities and enhance its efficiency.
Researchers at UC Santa Barbara led by biologist Chris Richardson have contributed to this growing field by developing a method that improves the efficiency of CRISPR/Cas9 editing. Importantly, their technique achieves this without the need for viral material to deliver the genetic template used in editing the target genetic sequence. The team’s findings were published in the prestigious journal Nature Biotechnology. They report that their approach increases the effectiveness of homology-directed repair, a crucial step in the gene editing process, by approximately threefold. Remarkably, this improvement is achieved without raising mutation frequencies or altering end-joining repair outcomes.
“We have identified a chemical modification that enhances non-viral gene editing and have also made an intriguing discovery about a novel type of DNA repair,” stated Richardson.
Find, cut and paste
The CRISPR/Cas9 method utilizes a defense mechanism employed by bacteria against viruses. Bacteria cut a fragment of the viral genetic material and incorporate it into their own DNA to recognize and destroy the virus in case of reinfection.
In gene editing, the Cas9 enzyme acts as molecular “scissors,” guided by the CRISPR system to snip specific genetic sequences. This creates an opportunity to replace the severed genes with improved versions, using the cell’s natural repair mechanisms. Successful editing leads to modified gene expressions and functions.
Typically, viral vectors are used to deliver the repair template DNA into the cell nucleus, where the genetic material resides. However, viral workflows are costly, difficult to scale, and potentially harmful to cells.
In contrast, nonviral templates offer advantages in terms of cost and scalability, but researchers still face challenges related to efficiency and toxicity. The Richardson Lab’s study discovered that introducing interstrand crosslinks into the workflow significantly enhanced homology-directed repair.
“We have observed approximately a threefold improvement in every workflow we have implemented using this approach,” explained Richardson.
Interstrand crosslinks refer to lesions that hold the two strands of a DNA helix together, preventing replication. Cancer chemotherapies utilize this mechanism to disrupt tumor growth and eliminate cancer cells. Interestingly, when these crosslinks were introduced into a homology-directed repair template, they stimulated the cell’s natural repair mechanisms and increased the likelihood of successful gene editing.
Overall, the Richardson Lab’s findings highlight the use of interstrand crosslinks as a strategy to boost gene editing efficiency without relying on viral vectors.
In essence, the researchers intentionally damaged the template DNA used in gene editing, expecting it to negatively affect the editing process. However, they were pleasantly surprised to discover that the damaged DNA actually resulted in a highly efficient and low-error nonviral gene editing system.
The serendipitous nature of this discovery is reminiscent of many scientific breakthroughs. During their experiments to purify proteins for DNA repair studies, lead author Hannah Ghasemi, a graduate student researcher, noticed unexpected changes in the outcomes.
“We introduced chemical modifications to the DNA templates to extract them from cells and examine the proteins bound to them. I was just checking if this modification had any impact on the editing process,” Ghasemi explained. “I expected either no change or even a negative effect on editing.”
Contrary to her expectations, Ghasemi observed a positive effect, with up to three times the editing activity compared to the uncrosslinked controls. Additionally, the team found that despite the increased editing, there was no rise in mutation frequency. While the specific mechanisms behind this result are still under investigation, the researchers have some hypotheses.
The researchers propose that when the cell detects the damaged DNA and attempts to repair it, the cell’s normal recombination process is delayed beyond its usual checkpoint. This extended time frame increases the likelihood that the editing process will be successfully completed. Understanding this novel process could provide insights into how cells recognize and accept editing reagents.
The team anticipates that this method will primarily be useful in ex-vivo gene editing applications, particularly in disease research and preclinical studies. Ghasemi explains that it allows for more effective gene knockdown and insertion in genomes, facilitating the study of systems outside the human body in a laboratory setting. This advancement enables the construction of disease models and testing of hypotheses related to disease mechanisms, potentially leading to improved clinical and therapeutic approaches.