Researchers have found that splitting the gene editor used in traditional CRISPR technology creates a more precise tool that can be switched on and off, with significantly less chance of causing unintended genome mutations. They say their novel tool can potentially correct around half of the mutations that cause disease.
CRISPR is one of those scientific terms that has made it into the everyday lexicon. Arguably one of the biggest discoveries of the 21st century, the gene-editing tool has revolutionized research and the treatment of genetic and non-genetic diseases. But the primary risk associated with CRISPR technology is ‘off-target edits,’ namely unexpected, unwanted, or even adverse alterations at locations in the genome other than the targeted site.
Now, researchers at Rice University have developed a new CRISPR-based gene-editing tool that’s more precise and significantly reduces the likelihood of off-target edits occurring.
“Our team set out to create a much-improved version that can be turned on or off as needed, providing an unparalleled level of safety and accuracy,” said Hongzhi Zeng, the study’s lead author. “This tool has the potential to correct nearly half of the disease-causing point mutations in our genome. However, current adenine base editors are in a constant ‘on’ state, which could lead to unwanted genome changes alongside the desired correction in the host genome.”
DNA consists of two linked strands that wind around each other, forming a double helix that resembles a twisted ladder. The ‘rungs’ of the ladder are made of base pairs, two complementary nucleotide bases held together by hydrogen bonds: adenine (A) pairs with thymine (T) and cytosine (C) with guanine (G).
Base pair mutations are also called ‘point mutations’ and are responsible for causing thousands of diseases. Traditional CRISPR uses either an adenine base editor (ABE) or cytosine base editor (CBE) to create point mutations at desired sites. Here, the researchers took an ABE and modified it.
They split the ABE into two separate proteins that remain inactive until a sirolimus molecule is added. Sirolimus, also known as rapamycin, is a drug with anti-tumor and immunosuppressant properties that’s used to prevent rejection in organ transplantation and treat certain types of cancer.
“Upon introduction of this small molecule, the two separate inactive fragments of the adenine base editor are glued together and rendered active,” said Zeng. “As the body metabolizes the rapamycin, the two fragments disjoin, deactivating the system.”
The researchers found that their novel split gene-editing tool had benefits in addition to remaining active for a shorter period of time than the original, intact ABE.
“Compared to an intact [base] editor, our version reduces off-target edits by over 70% and increases the accuracy of on-target edits,” Zeng said.
They tested their method by targeting the PCSK9 gene in a mouse liver. The PCSK9 gene makes a protein that helps regulate the amount of cholesterol in the bloodstream, so it’s therapeutically relevant for humans. Packaging their rapamycin-activated split ABE into an adeno-associated virus (AAV) vector, they found that it converted a single A●T base pair to a G●C base pair on the gene. This conversion is particularly useful as mutations in which G●C is mutated to an A●T base pair account for almost 50% of single-point mutations associated with human genetic diseases.
“We hope to see the eventual application of our split genome-editing tool with higher precision to address human health-related questions in a much safer way,” said Xue Gao, corresponding author of the study.
The study was published in the journal Nature Communications.
Source: Rice University
