CRISPR-Cas9 gene editing technology is widely used to to help study genes of interest and modify disease-associated genes. However, the system is associated with adverse effects, including mutations and potential toxicity, and so a way of reducing these unwanted effects will be needed to improve the utility of CRISPR technology in industry and in medicine. Researchers at Kyushu University and at Nagoya University School of Medicine have now developed an optimized genome-editing method that utilizes what they call a “safeguard single-guide RNA” to vastly reduce the risk of mutations and potentially enable more effective treatment of genetic diseases with much less risk of off target effects.

“In particular, we believe that this technology can make a significant contribution to the medical field,” said assistant professor Masaki Kawamata, PhD, at Kyushu University, who is first author and co-corresponding author of the team’s published paper in Nature Biomedical Engineering, titled “Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs,” in which they concluded, “The safeguard-sgRNA strategy may improve the safety and applicability of genome editing.”

CRISPR-Cas9 genome editing has revolutionized the food and medicine industries. The method involves introducing Cas9 nuclease—an enzyme that cuts DNA—into cells, with a synthetic guide RNA (gRNA) that directs the enzyme to the required location. The ability to cut the genome in this way gives scientists a method for deleting unwanted genes and adding new (functional) genes easily and quickly. And, as the authors wrote, “The precise regulation of the activity of Cas9 is crucial for safe and efficient editing.”

However, one of the drawbacks of genome editing is the growing concerns about the creation of off target effects and mutations. These problems can be caused by the enzyme targeting genomic sites that have a sequence similar to the target site. Similarly, mutations at the chromosome level can occur when genes are altered. “… in addition to well-known off-target effects, recent studies have documented several prevalent adverse effects of the standard CRISPR-Cas9 system in mammalian cells, including frequent p53 activation, cytotoxicity with severe DNA damage, large on-target genomic deletion and chromosomal rearrangement,” the team said. Safety issues have hindered clinical trials of gene therapy for cancer and the death of a patient undergoing treatment for muscular dystrophy was previously reported.

“Considering the potent activity of the current CRISPR-Cas9 and its frequent adverse effects, controlled inhibition of its activity would be a straightforward and powerful approach to improve its safety,” the researchers further noted. Various approaches have already been reported, they continued. “For this purpose, various options (for example, anti-Cas9 proteins, small-molecule inhibitors and oligonucleotides) have been demonstrated to limit Cas9 activity.”

Kawamata, together with co-corresponding author Hiroshi Suzuki, PhD, at the Nagoya University Graduate School of Medicine, and colleagues, hypothesized that current editing protocols that use Cas9 cause excessive DNA cleavage, resulting in some mutations effects. To test their hypothesis the researchers constructed a system called “allele-specific indel monitor system (AIMS)” in mouse cells, to evaluate the activity of Cas9 separately for each chromosome. “To determine the feasibility of Cas9 inhibition approaches, it is important to precisely determine the relationships among Cas9 activity strength, allelic configurations for editing, adverse effects and editing outcomes.,” they wrote. “… we developed a convenient but accurate experimental system to visualize genome editing dynamics, including large genomic deletions, in each allele at the single-cell level in living cells. This allele-specific indel monitor system (AIMS) allows the rapid and real-time quantitation of various editing patterns of a pair of alleles in a large number of clones without sequencing analysis.

The results of their tests using the AIMS technology showed that the commonly used CRISPR-Cas9 technology was associated with very high editing activity. They found that this high activity was causing some of the unwanted adverse effects, so they searched for a way to modify the gRNA as an approach to reduce the activity of the Cas9 enzyme. They found that an extra cytosine extension to the 5′ end of the gRNA was effective as a “safeguard” for the overactivity and allowed control over DNA cleavage. They called this fine-tuning system ‘safeguard gRNA’ ([C]gRNA).”

Their results were striking. Experiments showed that using the new technique, off-target effects and cytotoxicity were reduced, the efficiency of single-allele selective editing was increased, and the efficiency of homology-directed repair (HDR), the most commonly employed mechanism for DNA double-strand break repair, was enhanced.

To test the effectiveness of their approach in a medically relevant setting, the scientists then investigated a rare disease called fibrodysplasia ossificans progressiva. Using a mouse model, they were able to create the same gene mutation as that responsible for the human disease. Then, using patient-derived induced pluripotent stem (iPS) cells, they were able to precisely repair damage down to a single nucleotide specifically in the disease-associated allele causing the disease. This, they said, demonstrated that their technique could represent a safe and efficient approach for gene therapy. “Short cytosine extensions reduced p53 activation and cytotoxicity in human pluripotent stem cells, and enhanced homology-directed repair while maintaining bi-allelic editing,” they explained. “Longer extensions further decreased on-target activity yet improved the specificity and precision of mono-allelic editing.”

The team also constructed the first mathematical model of the correlation between various genome-editing patterns and Cas9 activity, which would enable the user to simulate the results of genome editing in an entire cell population. This breakthrough would allow researchers to determine the Cas9 activity that maximizes efficiency, reducing the enormous costs and labor required. “We also developed computational simulations to obtain an overall snapshot of the relationships among gRNA modification, Cas9 activity, Cas9 specificity, cytotoxicity and HDR efficiency,” they stated.

The results of their study, they claimed, “establish distinct optimal windows of Cas9 activity for diverse applications, including safe bi-allelic editing, mono-allelic editing, and HDR-based generation and correction of disease-associated single-nucleotide substitutions free from p53 activation.”

“Suzuki added, “We established a new genome editing platform that can maximize 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. 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.” In their paper the authors concluded, “Our comparison of ‘safeguard sgRNAs’, anti-CRISPR proteins and small-molecule inhibitors suggests that [C] extension is a convenient and safe tool.”

The group is now working on a start-up business plan to spread the new genome editing platform. “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,” Kawamata commented.

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