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A genome-altering method referred to as prime editing shows promise for addressing numerous ailments by converting defective genes into operative ones. Nevertheless, this technique comes with a slight risk of introducing inaccuracies that could be detrimental.
Researchers at MIT have recently discovered a method to significantly reduce the error rate of prime editing by utilizing altered versions of the proteins that play a role in the procedure. This breakthrough could facilitate the development of gene therapy options for a range of conditions, according to the researchers.
“This publication presents a novel strategy for gene editing that simplifies the delivery mechanism and avoids adding extra steps, while achieving a much more accurate edit with fewer unintended mutations,” states Phillip Sharp, an MIT Institute Professor Emeritus, a member of MIT’s Koch Institute for Integrative Cancer Research, and one of the lead authors of the latest research.
Utilizing their new method, the MIT team improved the error rate of prime editors from approximately one mistake in seven edits to one in 101 for the most commonly used editing mode, and from one error in 122 edits to one in 543 for a high-precision mode.
“For any medication, the goal is to be effective while minimizing side effects,” remarks Robert Langer, the David H. Koch Institute Professor at MIT, a member of the Koch Institute, and one of the lead authors of the current study. “In any situation where genome editing is applicable, I believe this approach will ultimately be a safer, superior option.”
Koch Institute research scientist Vikash Chauhan is the principal author of the study, which is published today in Nature.
The risk of inaccuracies
The initial iterations of gene therapy, first examined in the 1990s, focused on delivering new genes via viruses. Later, gene-editing methodologies employing enzymes such as zinc finger nucleases for gene correction were established. However, engineering these nucleases can be challenging, making their adaptation to target various DNA sequences a very time-consuming endeavor.
Years later, scientists discovered the CRISPR genome-editing system in bacteria, which provides a potentially simpler method for genome alteration. The CRISPR system includes an enzyme called Cas9 that can cleave double-stranded DNA at specific locations, alongside a guide RNA that directs Cas9 where to make the cut. Researchers have modified this technique to excise defective gene sequences or to introduce new ones, guided by an RNA template.
In 2019, experts at the Broad Institute of MIT and Harvard announced the creation of prime editing: an innovative system based on CRISPR that offers greater precision and reduced off-target effects. A recent investigation revealed that prime editors were effectively utilized to treat a patient with chronic granulomatous disease (CGD), a rare genetic disorder impacting white blood cells.
“Theoretically, this technology could one day be utilized to remedy hundreds of genetic disorders by correcting minor mutations directly in cells and tissues,” Chauhan notes.
One notable benefit of prime editing is that it does not necessitate a double-stranded cut in the target DNA. Instead, it employs a modified Cas9 that cleaves just one of the complementary strands, creating an opening where a new sequence can be inserted. A guide RNA delivered with the prime editor acts as the template for this new sequence.
However, once the new sequence is formed, it must compete with the original DNA strand to integrate into the genome. If the original strand prevails over the new one, the extra flap of new DNA may incorrectly integrate elsewhere, resulting in inaccuracies.
While many of these inaccuracies may be relatively innocuous, some could potentially lead to tumor formation or other complications. With the latest iteration of prime editors, the error rate varies from one in seven edits to one in 121 edits across different editing modes.
“The technologies available now are significantly more advanced than past gene therapy tools, yet there’s always a possibility for these unintended outcomes,” Chauhan expresses.
Accurate editing
To minimize those error rates, the MIT team opted to leverage a phenomenon they identified in a 2023 study. In that research, they discovered that while Cas9 typically cleaves at the same DNA site consistently, certain mutated forms of the protein exhibited a relaxation of these constraints. Instead of always cutting at the same position, these Cas9 variants would sometimes make their cuts one or two bases further along the DNA strand.
This flexibility, the researchers found, destabilizes the old DNA strands, enabling them to degrade and allowing the new strands to integrate without introducing errors more readily.
In the current study, the researchers successfully identified mutations in Cas9 that reduced the error rate to 1/20th of its original figure. By combining pairs of these mutations, they engineered a Cas9 editor that further decreased the error rate to 1/36th of the original level.
To enhance the accuracy of the editors, the researchers integrated their new Cas9 proteins into a prime editing framework that includes an RNA binding protein, which stabilizes the ends of the RNA template more efficiently. This final editor, referred to as vPE by the researchers, achieved an error rate of just 1/60th of the original, fluctuating from one in 101 edits to one in 543 edits across different editing modes. These evaluations were conducted in mouse and human cells.
The MIT team is actively focused on further enhancing the efficiency of prime editors through additional modifications of Cas9 and the RNA template. They are also exploring strategies to deliver the editors to specific body tissues, which has historically been a significant hurdle in gene therapy.
They anticipate that other research laboratories will begin adopting the new prime editing methodology in their studies. Prime editors are routinely employed to investigate various questions, including tissue development, the evolution of cancer cell populations, and cellular responses to drug treatments.
“Genome editors are extensively utilized in research laboratories,” Chauhan remarks. “While the therapeutic potential is thrilling, we are genuinely eager to observe how researchers begin to incorporate our editors into their research processes.”
The research received funding from the Life Sciences Research Foundation, the National Institute of Biomedical Imaging and Bioengineering, the National Cancer Institute, and the Koch Institute Support (core) Grant from the National Cancer Institute.
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