Advances in Gene Editing: Smaller Enzyme Yields Increased Efficacy for Therapeutic Applications

A recently developed gene-editing instrument, known as AsCas12f, presents a smaller alternative to the conventional Cas9 enzyme, while maintaining or even enhancing effectiveness in the treatment of genetic diseases. The modified enzyme has demonstrated promising results in mouse trials and holds potential for more streamlined and efficacious genome-editing approaches in humans.

The newly engineered CRISPR enzyme provides a more compact option for DNA editing without sacrificing the efficacy of existing instruments, thereby potentially enhancing patient care.

Researchers have unveiled a new gene-editing tool based on CRISPR technology that could revolutionize treatments for genetic disorders. This new enzyme, AsCas12f, is a modified version that is approximately one-third the size of the traditionally utilized Cas9 enzyme. Its reduced dimensions allow for a greater concentration to be delivered into living cells via carrier viruses, thereby increasing its overall efficacy.

To optimize the enzyme’s capabilities, scientists generated an assortment of possible AsCas12f mutations. Selected mutations were then combined to produce an AsCas12f enzyme with tenfold greater editing capabilities compared to its original, unmodified form. Trials in mice have already yielded successful outcomes, indicating promising prospects for more effective treatments in human healthcare.

Cryogenic electron microscopy was employed by the researchers to closely examine AsCas12f’s structure. A Differential Scanning Fluorimetry (DMS) “heatmap” was used to gauge how each individual mutation affected genome-editing efficiency. Darker shades of blue on the heatmap correspond to less favorable mutations, while darker shades of red suggest beneficial changes.

CRISPR technology has previously enabled scientists to make transformative changes to DNA, ranging from creating disease-resistant mosquitoes to augmenting the nutritional profile of food crops. Additionally, human trials have been initiated to tackle some of the most daunting health conditions. The magnitude of CRISPR’s potential impact was recognized in 2020, when Jennifer Doudna and Emmanuelle Charpentier were awarded the Nobel Prize in Chemistry for developing the highly accurate CRISPR-Cas9 tool.

Nonetheless, Cas9 has inherent limitations. The primary method for transferring genetic material into host cells involves the use of adeno-associated viruses (AAVs), which are generally safe for patients and have a lower risk of triggering unwanted immune reactions. Cas9’s relatively large size, however, approaches the maximum capacity of these AAVs, necessitating the search for a more compact alternative.

Professor Osamu Nureki from the University of Tokyo’s Department of Biological Sciences elucidated that the large dimensions of Cas9 often restrict its efficiency in gene therapy applications. Thus, a comprehensive, multi-institutional research effort was initiated to develop a smaller yet equally active Cas enzyme. They opted for AsCas12f, sourced from the bacteria Axidibacillus sulfuroxidans, which is less than one-third the size of Cas9 but previously demonstrated minimal activity in human cells.

Deep mutational scanning was employed to create a library of potential AsCas12f mutations, which led to the identification of over 200 mutations that increased its genome-editing effectiveness. These selected mutations were then engineered into a modified version of AsCas12f, resulting in an enzyme with genome-editing activity that is more than ten times higher than its original form and comparable to Cas9, but at a substantially reduced size.

Animal tests have already been carried out using this engineered enzyme, revealing its potential utility in human gene therapies for conditions like hemophilia, a disorder that impairs the blood’s ability to clot.

The research team acknowledges that their selection of mutations may not be the most optimal among all possible combinations. Future studies may leverage computational modeling or machine learning to refine the selection process further.

Professor Nureki stated that elevating the genome-editing efficacy of AsCas12f to a level comparable to Cas9 marks a significant milestone. The engineered enzyme could serve as the basis for more compact and effective gene-editing tools, thereby realizing the full therapeutic potential of gene therapy for treating genetic disorders.

Reference: “An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis,” authored by Tomohiro Hino et al., was published in the journal Cell on September 29, 2023. The study was funded by various organizations, including the Research Council of Lithuania, the Japan Foundation for Applied Enzymology, AMED, and the Cabinet Office, Government of Japan, among others.

Frequently Asked Questions (FAQs) about CRISPR AsCas12f

More about CRISPR AsCas12f

• CRISPR Technology Overview
• Gene Editing and CRISPR
• Introduction to Genetic Disorders
• Cryogenic Electron Microscopy in Molecular Biology
• Deep Mutational Scanning
• Adeno-Associated Viruses (AAVs) in Gene Therapy
• Nobel Prize in Chemistry 2020: CRISPR-Cas9
• Research Council of Lithuania
• Japan Foundation for Applied Enzymology
• AMED: Japan Agency for Medical Research and Development
• Cabinet Office, Government of Japan