Researchers have found that taking apart the gene editor used in traditional CRISPR technology can create a more precise tool that can be turned on and off, with a much lower chance of causing unintended genome mutations. They say their new tool has the potential to correct about half of all disease-causing mutations.
CRISPR is one of the scientific terms that has entered everyday vocabulary. This gene-editing tool is arguably one of the biggest discoveries of the 21st century, revolutionizing the research and treatment of genetic and non-genetic diseases. But the main risk associated with CRISPR technology is "off-target editing", which is the occurrence of unexpected, unnecessary or even adverse changes in the genome other than the target site.
Now, researchers at Rice University have developed a new gene-editing tool based on CRISPR technology that is more precise and greatly reduces the possibility of off-target editing.
"Our team set out to develop an improved version of the gene-editing tool that could be turned on or off as needed, providing unparalleled safety and accuracy," said Hongzhi Zeng, the study's first author. "Such a 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 constantly 'on' state, which can lead to unwanted genomic changes while making the desired corrections in the host genome."
DNA is made up of two connected strands that wrap around each other to form a double helix that resembles a twisted ladder. The "rungs" of the ladder are made up of base pairs, which are two complementary nucleotide bases held together by hydrogen bonds: adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).
Base pair mutations, also known as "point mutations," are responsible for thousands of diseases. Traditional CRISPR uses an adenine base editor (ABE) or a cytosine base editor (CBE) to create point mutations at the desired site. Here, the researchers used ABE and modified it.
They separated ABE into two separate proteins, which remained inactive until the sirolimus molecule was added. Sirolimus, also known as rapamycin, is a drug with anti-tumor and immunosuppressive properties used to prevent rejection in organ transplants and to treat certain types of cancer.
"When this small molecule is introduced, two independent inactive segments of the adenine base editor are glued together and become active," Zeng said. "When the body metabolizes rapamycin, these two fragments separate, rendering the system inactive."
The researchers found that in addition to remaining active for a shorter period of time than the original, intact ABE, their new split gene-editing tool had other benefits.
"Compared to the full [base] editor, our version reduces off-target edits by more than 70 percent and improves the accuracy of on-target edits," Zeng said.
They tested their approach by targeting the PCSK9 gene in mouse livers. The PCSK9 gene makes a protein that helps regulate cholesterol levels in the blood and therefore has therapeutic implications for humans. They packaged the rapamycin-activated split ABE into an adeno-associated virus (AAV) vector and found that it could convert a single A●T base pair on the gene into a G●C base pair. This conversion is particularly useful because mutations of the G●C to A●T base pair account for almost 50% of single-point mutations associated with human genetic diseases.
"We hope to see our split genome editing tools eventually be applied to solve human health-related problems with greater precision," said Gao Xue, the study's corresponding author.
The research was published in the journal Nature Communications.