CRISPR-Cas9 is widely used for genome editing by studying genes of interest and modifying disease-associated genes. However, this process can be associated with potential side effects, including unwanted mutations and toxicity.
Researchers at Kyushu University and Nagoya University School of Medicine in Japan have developed an optimal method of genome editing that greatly reduces mutations, opening the door to more effective treatment of genetic diseases with fewer unwanted mutations.
Their findings are published in Nature Biomedical Engineering.
CRISPR-Cas9-centered genome editing technology has revolutionized the food and drug industry. In the technology, Cas9 nuclease, an enzyme that cuts DNA, is inserted into cells with synthetic guide RNA (gRNA) that guides the enzyme to its desired location. By cutting the genome, unwanted genes can be removed, and new (functional) genes can be 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 that have sequences similar to those of the target site. Likewise, mutations at the chromosomal level can occur when genes are altered, which hinders gene therapy clinical trials. The researchers hypothesize that current editing protocols using Cas9 lead to excessive DNA cleaving, resulting in multiple mutations.
Test gene editing
To test this hypothesis, a group consisting of Masaki Kawamata at Kyushu University and Hiroshi Suzuki at the Nagoya University School of Medicine built a system called “AIMS” in mouse cells, which evaluates Cas9 activity separately for each chromosome. The results show that commonly used methods are associated with very high editing activity. They determined that this high activity caused several unwanted side effects, so they looked for gRNA modification methods that could suppress it.
They found that extra cytosine extension to the 5′ end of the gRNA was effective as a “shield” against overactivity and allowed control over DNA cleavage. They called this refinement system ‘safeguard gRNA’ ((C)gRNA).
Using the new technique, off-target effects and cytotoxicity are reduced, the efficiency of single allele selective editing is increased, and the efficiency of homology-directed repair, the most commonly used mechanism for DNA double-strand damage repair, is improved.
To test its effectiveness in a medical setting, they investigated a rare disease called fibrodysplasia ossificans progressiva. Using a mouse model, they created the same genotype as the human version of the disease. Then, using patient-derived iPS cells, they were able to precisely repair defects down to one nucleotide specifically in disease-associated alleles that cause disease, demonstrating this technique as a safe and efficient method of gene therapy.
The team also built the first mathematical model of the correlation between various patterns of genome editing and Cas9 activity, which will allow users to simulate the results of genome editing across cell populations. This breakthrough will enable researchers to define Cas9 activities that maximize efficiency, reducing costs and manpower required.
Application in medicine
“We created a new genome editing platform that can maximize desired editing efficiency by developing (C)gRNA activity regulators with suitable Cas9 activity. In addition, we found that ‘gRNA safeguards’ can be applied to various CRISPR tools that require gRNA by regulating their activity, such as those using Cas12a, which has a different DNA cleavage mechanism,” says 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 useless and even harmful to cells. Controlling expression levels by (C)gRNA is an important technology that can be used for a variety of applications, including the implementation of precise gene therapy.”
The group is now working on an initial business plan to deploy the new genome editing platform.
“In particular, we believe that this technology can make a significant contribution in the medical field,” said Kawamata.
“We are currently evaluating its therapeutic efficacy and safety for selected target diseases in cell and animal trials and using it to help develop therapeutic drugs and gene therapy methods, especially for rare diseases for which treatment methods have not been established.”
The discovery comes after scientists at the Helmholtz Institute Würzburg in Germany, and Benson Hill, Inc. (Missouri) and Utah State University in the US discovered a nuclease, which they named Cas12a2, that represents an entirely new type of CRISPR immune defense.
Unlike other previously known nucleases of the immune system CRISPR-Cas, the source of the ‘gene scissors’, Cas12a2 destroys DNA to kill infected cells. These findings could lead to new CRISPR technology.