Newswise — (Santa Barbara, Calif.) — Genetic modification is a potent technique for both investigation and treatment. Following the development of the Nobel Prize-winning CRISPR/Cas9 approach, a precise and fast instrument for genome editing identified in 2012, researchers have been striving to investigate its potential and enhance its efficiency.
A technique has been developed by scientists in the lab of UC Santa Barbara biologist Chris Richardson that expands the toolkit for gene editing. The method enhances the effectiveness of CRISPR/Cas9 editing without employing viral agents to distribute the genetic template utilized to modify the designated genetic sequence. In their recent publication in the journal Nature Biotechnology, the researchers noted that their method stimulates homology-directed repair, a critical stage in the gene editing process, by about three times "without raising mutation rates or modifying end-joining repair results."
Richardson commented, "We have identified a chemical alteration that enhances gene editing without the use of viral vectors and also identified a fascinating novel form of DNA repair."
Find, Cut and Paste
The CRISPR/Cas9 technique exploits a defense mechanism utilized by bacteria against viral invaders. Bacteria cut out a segment of the virus's genetic material and insert it into their own genome, enabling them to recognize and eliminate the invader if it infects them again.
In gene editing, this mechanism employs the Cas9 enzyme as molecular "scissors" to sever sequences that it recognizes, guided by the CRISPR system. This incision presents an opportunity to substitute the cut genes with comparable (homologous) but enhanced ones, using the cell's innate repair processes. If successful, the cell's expressions and functions can be modified accordingly.
To transport the repair template DNA to the cell nucleus, where the genetic material is located, viruses are frequently utilized. Despite being efficient, viral methods are "costly, hard to expand, and have the potential to be harmful to cells," the scientists explain.
Richardson stated, "In every workflow that we have employed this technique, we have observed an improvement in efficiency of approximately three times."
Interstrand crosslinks are anomalies that hold the two strands of a DNA helix together, rendering them incapable of replication. This mechanism is used by cancer chemotherapies to disrupt tumor growth and eliminate cancer cells. However, when added to a homology-directed repair template, these crosslinks were discovered to activate the cell's innate repair mechanisms, increasing the chances of successful gene editing.
"What we've essentially done is taken this template DNA and impaired it," Richardson explained. "In reality, we've impaired it in the most severe manner possible. However, the cell doesn't identify it as garbage and discard it. Rather, the cell acknowledges it as useful and integrates it into its genome." The outcome is a highly effective and low-error nonviral gene editing system.
As with many scientific breakthroughs, their discovery was somewhat fortuitous. While attempting to purify proteins to research DNA repair, lead author and graduate student researcher Hannah Ghasemi observed unexpected changes in the results of their experiments.
"We were adding these chemical modifications to the DNA templates so that we could extract them from the cells and determine which proteins were binding to them, and I was simply examining whether this modification had any impact on the editing," she explained. "I anticipated seeing either no difference or even a negative impact on the editing."
Instead, she discovered a positive effect, with up to three times the editing activity of the uncrosslinked controls. Additionally, the team discovered that despite the increase in edits and therefore the likelihood of errors, there was no increase in mutation frequency. They are still researching the exact mechanisms that led to this outcome, but they have some hypotheses.
Richardson believes that the cell detects and attempts to repair the damaged DNA that they added the crosslink to, causing the cell to delay past a checkpoint where it would normally stop the recombination process. By prolonging the amount of time it takes for the cell to complete this recombination, it increases the likelihood that the edits will be successful. He also suggests that studying this new process could lead to a better understanding of how cells detect editing reagents and how they decide whether to accept them or not.
According to the research team, this technique will be most useful for gene editing applications done outside of a living organism (ex-vivo), such as in disease research and preclinical studies.
According to Ghasemi, this method will be most useful for ex-vivo gene editing applications in disease research and preclinical studies. It enables more effective gene knockdown and genome insertion in lab settings, allowing for the efficient building of disease models and testing of hypotheses. This development has the potential to lead to improved clinical and therapeutic approaches.
RSS