Improvement of Ultra-small Cas12m Lacking Self-cleavage Activity Opens Path for Chronic Hepatitis B Treatment via Single AAV Delivery
Background
Epigenetic editing using CRISPR gene-editing technology is gaining attention as a therapeutic strategy that precisely regulates specific gene expression without altering the DNA sequence itself. This advantage stems from its ability to fundamentally eliminate risks such as chromosomal abnormalities and off-target cleavage that arise when directly cutting the DNA double helix. The existing system has mainly utilized dCas9 (dead Cas9) complexes, in which the catalytic site of the Cas9 protein derived from Streptococcus pyogenes is mutated. This approach involves fusing transcriptional repressor factors or DNA methyltransferases to silence specific gene expression.
The problem lies in the massive molecular size of dCas9, which spans 1,368 amino acids. It exceeds the effective loading limit of 4.7 kilobases (kb) of the adeno-associated virus (AAV), the most preferred in vivo gene delivery vector. While the dual AAV method, which splits the system into two parts for delivery, was proposed as an alternative, it failed to overcome hurdles such as decreased simultaneous infection efficiency in cells and high immunogenicity. Therefore, there was an urgent need to discover ultra-small binding proteins that could be loaded into a single AAV capsid along with therapeutic transcriptional regulation modules.
Key Discoveries
The researchers identified PmCas12m, a new V-M subtype CRISPR protein, from the methanol-utilizing bacterium Pelomicrobium methylotrophicum by linking bioinformatic screening, structural prediction, and functional validation. It features flexible recognition of the protospacer adjacent motif (PAM), 5'-YTN-3', located adjacent to the protospacer. Even without artificial genetic manipulation, it exhibited biochemical characteristics of maintaining strong target binding affinity while being completely devoid of double-stranded DNA cleavage activity. This represents a fundamental distinction from existing dCas proteins, which required the forced inactivation of their catalytic activity.
Determining the 3D structure via cryo-electron microscopy (cryo-EM) provided a critical breakthrough in revealing the target binding mechanism at the atomic level. Using this structural data as a compass, researchers applied intensive deep mutational scanning (DMS) and protein engineering. As a result, they succeeded in developing xCas12m, an ultra-small variant with maximized target binding affinity and selectivity in human cells. The researchers designed the xCas12m-CRISPRoff system by connecting a DNA methylation silencing module to xCas12m and fully encapsulated it within a single AAV vector. Administration of a single AAV formulation to a mouse model infected with the hepatitis B virus (HBV) resulted in sustained silencing of viral replication pathways within hepatocytes. This achievement resulted in the long-term, significant inhibition of viral antigen expression with a single in vivo injection.
Significance and Outlook
This achievement has overcome the practical barriers to in vivo epigenetic therapy using a single viral vector by minimizing the size of the target-binding domain of the gene editor. A chronic condition for which a cure has been impossible with existing antivirals due to circular covalently closed DNA (cccDNA) hidden within the nuclei of hepatocytes. The xCas12m-CRISPRoff platform has demonstrated therapeutic potential for the long-term silencing of viral genes without cleaving host chromosomes. This is why expectations are high for expansion beyond infectious diseases to targets such as tumor suppressor genes and single-gene genetic disorders.
However, the hurdles to clinical entry remain a clear reality. Off-target silencing, where unintended methylation occurs in non-target regions of the human genome, must be continuously tracked at the whole-genome level. The control of neutralizing antibody formation and immune rejection responses against bacterial-derived proteins is also an essential evaluation criterion. If a long-term safety profile is established, it is expected to become the standard platform for developing single AAV-based epigenetic therapeutics.
CRISPR-based epigenome editing represents a programmable strategy to precisely modulate gene expression, holding promise for therapeutic applications. However, the large size of dCas proteins substantially impedes delivery using adeno-associated virus (AAV) vectors. Here, through iterative bioinformatics analysis, structure-guided predictions and functional assays, we identified and characterized a miniature subtype V-M CRISPR-Cas12m from Pelomicrobium methylotrophicum. PmCas12m exhibited flexible 5'-YTN-3' PAM-dependent recognition and robust double-stranded DNA-binding properties while lacking DNA cleavage activity, thus rendering it a valuable tool for epigenome editing. Cryo-electron microscopy structures of PmCas12m unveiled its molecular mechanism of target DNA binding. Guided by these structural insights, we used deep mutational scanning and protein engineering to develop xCas12m, a hypercompact variant with highly potent and specific epigenome-editing capabilities in human cells. We further constructed the xCas12m-CRISPRoff platform in a single AAV vector, which achieved durable epigenetic silencing and effective inhibition of hepatitis B virus infection in a mouse model. Collectively, these findings establish xCas12m as a versatile epigenome-editing platform with notable potential for treating diseases, paving the way for clinical translation of epigenetic therapies.
This research has increased the practical possibility of mass production and clinical application by simplifying epigenome editing technology, which previously relied on complex multi-vector delivery systems, into a single AAV formulation. In the pharmaceutical and biotech industries, the dual AAV process doubles production costs and makes quality control extremely difficult due to imbalances in viral delivery. The ability to load single AAVs enables simultaneous improvements in yield for Contract Development and Manufacturing Organization (CDMO) processes and reductions in drug prices.
In clinical settings, it offers a one-time gene therapy option for the purpose of a cure for patients with chronic hepatitis B, who faced a high risk of recurrence upon discontinuation of standard treatments. This is possible because a therapeutic mechanism has been established that permanently epigenetically silences only the target viral genes, without the risk of chromosomal deletions or cancer induction associated with conventional gene-editing tools. In the future, it holds great potential to drive the development of pipelines for refractory diseases with strictly limited AAV delivery capacities, such as ophthalmic conditions requiring local administration or rare degenerative central nervous system disorders, extending beyond liver diseases.