Optimized genome-editing methods open door to more effective treatments for genetic diseases

CRISPR-Cas9 is widely used to edit genomes by studying genes of interest and altering disease-associated genes. However, this process is accompanied by side effects such as unwanted mutations and toxicity. Therefore, new technologies that mitigate these side effects are needed to improve their industrial and medical utility. Currently, researchers at Kyushu University in southern Japan and Nagoya University School of Medicine in central Japan have developed an optimized genome-editing method that significantly reduces mutations, making it more effective for genetic diseases with fewer unwanted mutations. opened the door to new treatments. Their findings were published in Nature Biomedical Engineering.

Genome editing technology centered on CRISPR-Cas9 has revolutionized the food and pharmaceutical industries. In this technique, Cas9 nuclease, an enzyme that cleaves DNA, is introduced into cells along with a synthetic guide RNA (gRNA) that directs the enzyme to where it is needed. By cutting the genome, unwanted genes can be removed and new (functioning) genes added easily and quickly.

One of the drawbacks of genome editing is the growing concern about mutations and off-target effects. This is often caused by enzymes targeting genomic sites with sequence similarity to the target site. Similarly, alterations in genes can lead to mutations at the chromosomal level, hampering clinical trials of gene therapy for cancer and even leading to death in patients undergoing treatment for muscular dystrophy. The group hypothesized that current editing protocols using Cas9 cause excessive DNA breaks, resulting in some mutations.

In order to test this hypothesis, a group led by Assistant Professor Masaki Kawamata of Kyushu University and Professor Hiroshi Suzuki of the Nagoya University Graduate School of Medicine constructed a system called “AIMS” in mouse cells to detect Cas9 activity on each chromosome. I rated it. Their results showed that commonly used methods were associated with very high editorial activity. They determined that this high activity caused some of the unwanted side effects, so they looked for gRNA modifications that could suppress it. They found that an extra cytosine extension to the 5′ end of gRNAs is effective as an overactive ‘protection’ and allows control of DNA cleavage. They called this fine-tuning system “safeguard gRNA” ([C]gRNA)”.

Their results were astonishing. Using their new technology, off-target effects and cytotoxicity are reduced, and selective editing of single alleles is more efficient, the most commonly used mechanism for DNA double-strand break repair. Improved efficiency of homology-based repair.

To test its effectiveness in clinical practice, we investigated a rare disease called fibrodysplasia ossificans progressive. Using a mouse model, they were able to create genotypes identical to the human version of the disease. It was possible to precisely repair damage down to one nucleotide of , demonstrating the usefulness of the technique as a safe and efficient gene therapy method.

The team also built the first mathematical model of the correlation between different genome editing patterns and Cas9 activity. This allows users to simulate the consequences of genome editing across cell populations. This breakthrough will allow researchers to determine her Cas9 activity that maximizes efficiency, reducing the enormous cost and effort required.

By developing an activity regulation mechanism, we have established a new genome editing platform that can maximize the desired editing efficiency. [C]gRNA with appropriate Cas9 activity. Furthermore, we found that the ‘safeguard gRNA’ can be applied to various CRISPR tools that require gRNA by modulating the gRNA’s activity, such as tools that use Cas12a with different DNA cleavage mechanisms. In techniques that use Cas9 to activate or repress genes of interest, such as CRISPR activation and CRISPR interference, excessive induction or repression of gene expression can be futile and even harmful to cells.control of expression levels by [C]gRNA is an important technology that can be used for a variety of applications, including the delivery of precision gene therapy. ”

Professor Hiroshi Suzuki, Graduate School of Medicine, Nagoya University

The group is currently working on a start-up business plan to popularize the new genome editing platform. “In particular, we believe that this technology can greatly contribute to the medical field,” said Dr. Kawamata. “We are currently evaluating the treatment Effectiveness We are enhancing the safety of selected target diseases in cell and animal experiments and using them to help develop drugs and gene therapies, especially for rare diseases for which no cure exists. ”