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New Technique Opens the Door to Safer Gene Editing by Reducing the Mutation Problem in Gene Therapy

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CRISPR-Cas9 is widely used to edit the genome by studying genes of interest and modifying disease-associated genes. But this process is associated with side effects, including unwanted mutations and toxicity. Therefore, a new technology that reduces these side effects is needed to improve its usefulness in industry and medicine. Now, researchers at Kyushu University in southern Japan and Nagoya University School of Medicine in central Japan have developed an optimised genome-editing method that vastly reduces mutations, opening the door to more effective treatment of genetic diseases with fewer unwanted mutations. Their findings were published in Nature Biomedical Engineering.

Genome-editing technology centred on CRISPR-Cas9 has revolutionised the food and medicine industries. In this technology, Cas9 nuclease, an enzyme that cuts DNA, is introduced into the cell with a synthetic guide RNA (gRNA) that guides the enzyme to the required location. By cutting the genome, unwanted genes can be deleted, and new (functional) genes can be added in easily and quickly.

One of the drawbacks of genome editing is that there are growing concerns about mutations and off-target effects. The enzyme frequently causes this by targeting genomic sites with a sequence similar to the target site. In the same way, changing genes can cause mutations at the chromosome level. This has slowed down clinical trials of gene therapy for cancer and even killed people who were getting treatment for muscular dystrophy. The group hypothesised that current editing protocols that use Cas9 cause excessive DNA cleavage, resulting in some of the mutations.

To test this idea, Assistant Professor Masaki Kawamata from Kyushu University and Professor Hiroshi Suzuki from Nagoya University Graduate School of Medicine built a system in mouse cells called “AIMS” that checked the activity of Cas9 for each chromosome separately. Their results showed that the commonly used method was associated with very high editing activity. They determined that this high activity was causing some unwanted side effects, so they searched for gRNA modification methods that could suppress it. They discovered that adding an extra cytosine to the 5′ end of the gRNA worked as a “safeguard” against the overactivity and let them control the cutting of DNA. They called this fine-tuning system “safeguard gRNA” ([C]gRNA).

Their results were striking. With their new method, they were able to reduce off-target effects and cytotoxicity while increasing the effectiveness of single-allele selective editing and homology-directed repair, which is the most common way to fix DNA double-strand breaks.

To test its effectiveness in a medical setting, they investigated a rare disease called fibrodysplasia ossificans progressiva. Using a mouse model, they were able to create the same genotype as the human version of the disease. Then, using patient-derived iPS cells, they were able to precisely repair damage down to a single nucleotide, specifically in the disease-associated allele causing the disease, demonstrating their technique’s usefulness as a safe and efficient gene therapy method.

The team also made the first mathematical model of the relationship between different genome-editing patterns and Cas9 activity. This model would let someone simulate what would happen if genome editing was done to a whole population of cells. This breakthrough would allow researchers to determine the Cas9 activity that maximises efficiency, reducing the enormous costs and labour required.

“We established a new genome editing platform that can maximise the desired editing efficiency by developing activity-regulating [C]gRNAs with appropriate Cas9 activity. Furthermore, we found that ‘safeguard gRNA’ can be applied to various CRISPR tools that require gRNAs by regulating their activities, such as those using Cas12a, which has a different DNA cleavage mechanism,” said Professor Suzuki. “For techniques that use Cas9 to activate or repress genes of interest, such as CRISPR activation and CRISPR interference, excessive induction or suppression of gene expression may be not useful and even harmful to cells. Controlling expression levels by [C]gRNA is an important technology that can be used for various applications, including the implementation of precise gene therapy.”

The group is now working on a start-up business plan to spread the new genome editing platform. “In particular, we believe that this technology can make a significant contribution to the medical field,” said Dr Kawamata. “We are currently evaluating its therapeutic efficacy and safety for selected target diseases in cell and animal experiments and using it to help develop therapeutic drugs and gene therapy methods, especially for rare diseases for which no treatment methods have yet been established.”

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