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February 4, 2020

DNA Prime Editing: A New CRISPR-Based Method to Correct Most Disease-Causing Mutations

JAMA. 2020;323(5):405-406. doi:10.1001/jama.2019.21827

A new genome editing method may overcome critical barriers to correcting disease-causing genetic mutations. The approach draws on the popular clustered regularly interspaced short palindromic repeat (CRISPR)–associated 9 (Cas9) technology but avoids some of its undesired effects on DNA. In principle, the technique—called prime editing—could correct an estimated 89% of genetic variants known to be associated with human diseases.

The strategy, described recently in a study published in Nature, relies on prime editors—an altered form of the Cas9 protein and an RNA that together orchestrate a series of DNA targeting, writing, and repair steps that result in an edit. Unlike with typical CRISPR-Cas9 technology, the prime editor Cas9 doesn’t make double-stranded cuts in DNA, which can lead to uncontrolled DNA insertions and deletions at the cut site. Instead, the altered Cas9 only snips a single strand of the double helix.

Making double-stranded cuts can be useful for disrupting genes and for moving large segments of DNA, according to the study’s senior author, David Liu, PhD, of the Broad Institute of Harvard and MIT. But using double-stranded cuts to make precise DNA changes has proven more difficult.

Liu and his colleagues first overcame this hurdle in 2016, when they developed so-called base editors. Although base editing uses CRISPR’s targeting ability, it directly converts one nucleotide base into another instead of cutting the double helix. Base editors can efficiently correct 4 of the most common types of single-base mutations, while avoiding insertion and deletion byproducts, but they can’t fix all errors.

Prime editing goes a step further. Not only can it swap single DNA bases, but it can also make both deletions and insertions.

“If CRISPR-Cas9 is like scissors and base editors are like pencils, then prime editors are like word processors, capable of searching for target DNA sequences and precisely replacing them with edited DNA sequences,” Liu told JAMA. He noted that all 3 technologies have their own strengths and weaknesses and that each will likely have unique and useful roles in applications such as medicine and agriculture.

Liu and his team used prime editing to perform more than 175 edits in various types of human cells, including inserting new DNA segments up to 44 bases long and removing segments up to 80 bases long. Some of the edits hinted at future health applications: using human cells, the researchers successfully corrected the primary genetic causes of sickle cell disease and Tay-Sachs disease. The technique proved to be more efficient than traditional Cas9 editing, with less off-target editing at known Cas9 off-target sites.

A prime editor’s protein component fuses an altered form of the Cas9 enzyme, which cuts DNA, and a reverse transcriptase, which generates complementary DNA from an RNA template. The technique’s RNA component is a prime editing guide RNA (pegRNA) that specifies the targeted DNA site and encodes the desired edit.

The reverse transcriptase directly copies the part of the pegRNA that encodes the edited DNA sequence into the target site, resulting in a new flap of DNA that contains the edit. When the cell incorporates this edited flap, it replaces the original DNA sequence on both strands of the DNA double helix.

“The key to prime editing’s versatility is that the part of the pegRNA that specifies the edited DNA sequence can be virtually any sequence,” Liu said. This advantage allows the approach to install all possible point mutations, small insertions, small deletions, and their combinations into target DNA sites. The researchers corrected the Tay-Sachs mutation by removing a 4-base insertion, for example. In another edit, they deleted 2 nucleotide bases at a specific position in the human genome and converted 1 nearby base into another—a G to a T.

“In many respects, this first report of prime editing is the beginning, rather than the end, of a longstanding aspiration to be able to make any DNA change in any position of a living cell or organism, including human patients with genetic diseases,” Liu said. He noted that much work remains—including tests of different delivery methods to target diverse cell types—before this line of research can make its way into the clinic.

Samuel Sternberg, PhD, of Columbia University in New York City, said he sees considerable potential for the strategy. “There’s no doubt that prime editing offers promise for clinical applications to treat disease,” said Sternberg, who was part of the research team that discovered the CRISPR-Cas9 gene editing technology. “Numerous CRISPR-based technologies have already entered clinical trials, with early indications of efficacy, and prime editing will broaden the list of disease indications where genome editing offers therapeutic potential.”