A new precise gene-editing tool from MIT could transform medicine

Researchers at MIT have now discovered a way to dramatically reduce these errors by changing key proteins that drive the editing process. They believe this improvement could make gene therapy safer and more practical for treating a wide range of diseases.

“This paper outlines a new approach to performing gene editing that neither complicates the delivery system nor adds additional steps, but results in much more precise editing with fewer unwanted mutations,” says Philip Sharp, an MIT professor emeritus, a member of MIT’s Koch Institute for Integrative Cancer Research, and one of the senior authors of the new study.

Using their improved method, the MIT team reduced the rate of errors in initial editing from approximately one in seven edits to about one in 101 for the most common type of editing. In the more precise editing mode, the improvement went from one in 122 to one in 543.

“For any drug, what you want is something that is effective, but with as few side effects as possible,” says Robert Langer, the David H. Koch Institute Professor at MIT, a member of the Koch Institute, and one of the senior authors of the new study. “For any disease where genome editing might be done, I think this will ultimately be a safer and better way to do it.”

Koch Institute research scientist Vikash Chauhan led the study, which was recently published in nature.

Probability of error

In the 1990s, early gene therapy efforts relied on introducing new genes into cells using modified viruses. Later, scientists developed techniques that use enzymes such as zinc finger endonucleases to directly repair genes. These enzymes worked but were difficult to re-engineer for new DNA targets, making them slow and cumbersome to use.

The discovery of the CRISPR system in bacteria changed everything. CRISPR uses an enzyme called Cas9, guided by a piece of RNA, to cut DNA at a specific location. The researchers modified it to remove defective DNA sequences or insert corrected sequences using an RNA-based template, making gene editing faster and more flexible.

In 2019, scientists at the Broad Institute of MIT and Harvard University introduced prime editing, a new version of CRISPR that is more precise and less likely to affect unintended regions of the genome. More recently, prime editing was successfully used to treat a patient with chronic granulomatous disease (CGD), a rare disorder that weakens white blood cells.

“In principle, this technology could eventually be used to treat hundreds of genetic diseases by correcting small mutations directly in cells and tissues,” says Chauhan.

One advantage of prime editing is that it does not require a double-strand cut in the target DNA. Instead, it uses a modified version of Cas9 that cuts just one of the complementary strands, opening a flap where a new sequence can be inserted. The guide RNA delivered with the primer serves as a template for the new sequence.

One reason prime editing is considered safer is that it does not cut both strands of DNA. Instead, it makes a gentler, single-strand cut using a modified Cas9 enzyme. This opens a small flap in the DNA where a new corrected sequence can be inserted, guided by the RNA template.

Once the corrected sequence is added, it should replace the original DNA strand. If the old tape is reattached instead, the new part can sometimes end up in the wrong place, resulting in unintended errors.

Most of these errors are harmless, but in rare cases they can contribute to tumor growth or other health problems. In current elementary editing systems, the error rate can vary from about one in seven edits to one in 121, depending on the editing mode.

“The technologies we have now are much better than previous gene therapy tools, but there is always a chance of these unintended consequences,” says Chauhan.

Careful editing

To reduce these error rates, the MIT team decided to take advantage of a phenomenon they observed in a 2023 study. In that paper, they found that while Cas9 typically cuts the same DNA site every time, some mutant versions of the protein show a relaxation of those constraints. Instead of always cutting the same site, these Cas9 proteins sometimes cut one or two bases along the DNA sequence.

The researchers discovered that this relaxation makes old DNA strands less stable, and thus degrade, making it easier for new strands to integrate without any errors.

In the new study, the researchers were able to identify Cas9 mutations that reduced the error rate to 1/20 of its original value. Then, by combining pairs of those mutations, they created a Cas9 editor that reduced the error rate even further, to 1/36 of the original amount.

To make the editors more precise, the researchers incorporated the new Cas9 proteins into a master editing system containing an RNA-binding protein that stabilizes the ends of the RNA template more efficiently. This final editor, which the researchers call vPE, had an error rate of only 1/60 of the original, ranging from one in 101 edits to one in 543 edits for the different editing modes. These tests were performed on mice and human cells.

The MIT team is now working to improve the efficiency of the prime editors, through further modifications to Cas9 and the RNA template. They are also working on ways to deliver the editors to specific tissues of the body, which has been a long-standing challenge in gene therapy.

They also hope that other labs will begin using the new prime-editing approach in their research studies. Primers are typically used to explore many different questions, including how tissues develop, how cancer cell populations develop, and how cells respond to drug treatment.

“Genome editors are widely used in research laboratories,” says Chauhan. “So the therapeutic aspect is exciting, but we’re really excited to see how people start incorporating our editors into their research workflow.”

The research was funded by the Life Sciences Research Foundation, the National Institute of Biomedical Imaging and Bioengineering, the National Cancer Institute, and a Koch Institute Support Grant (core) from the National Cancer Institute.