Background

When treating diseases, instead of directly cutting and suturing damaged genes, a method that precisely turns on the expression switches of target genes is gaining attention. This is CRISPR activation (CRISPRa) technology, which combines transcriptional activators with Cas proteins that have had their cleavage activity removed. By not cutting DNA double strands, it reduces the risk of permanent sequence damage while restoring the production of deficient proteins to normal levels.

However, structural limitations were encountered during in vivo delivery. Adeno-Associated Virus (AAV), the most representative carrier for transporting therapeutic genes into cells, has a strict genome loading limit of approximately 4.7 kilobases (kb). The widely used SpdCas9 protein derived from Streptococcus pyogenes exceeds 4kb in size alone, making it impossible to package both the transcriptional activation factors and single guide RNA (sgRNA) into a single virus.

Dual-vector strategies that split the virus into two parts resulted in transduction efficiency dropping to less than half and increased risks of immune side effects. Although ultra-small Cas proteins have been developed to significantly reduce their size, the challenge of rapidly declining DNA binding affinity and transcriptional induction efficiency as molecular size decreased has persisted. Precise control of the simultaneous activation of multiple genes in complex disease mouse models has long remained an unfinished task.

Key Findings

An international research team identified Cas12m after screening for Cas12 enzymes in natural microorganisms. By precisely analyzing the three-dimensional structure of Cas12m and applying protein engineering techniques, they developed an improved variant with inactivated catalytic activity while significantly enhancing target binding affinity. The resulting platform is smCRISPRa, an ultra-small gene activation system.

The core enzyme of smCRISPRa is only about 750 amino acids in length, representing a volume roughly half that of the existing SpCas9 protein. This significant reduction in size enables the compact packaging of not only the enzyme-coding sequence but also powerful transcriptional activation domains and multiple guide RNA expression cassettes within a single 4.7 kb AAV vector.

The researchers also preserved the inherent crRNA self-cleavage capability of Cas12m. Without the need for complex multiple promoters, the enzyme itself precisely cleaves individual guide RNAs from crRNA precursors linked in series. As a result, multiplexed activation—simultaneously stimulating multiple target genes using a single delivery vector—was stably achieved in vivo.

The research team verified therapeutic efficacy using the mdx mouse model, a model for the rare genetic disease Duchenne Muscular Dystrophy (DMD). They chose an indirect strategy that does not directly manipulate the mutated dystrophin gene. The method involves simultaneously activating the utrophin gene, which can substitute for the function of the deficient protein, and the follistatin gene, which aids muscle regeneration.

In the skeletal muscle of mdx mice injected with smCRISPRa via a single AAV, the mRNA transcription levels of both genes increased significantly compared to the control group. Serum creatine kinase levels, which indicate muscle cell membrane damage, decreased markedly, and muscle strength showed a pattern of recovery close to normal levels in grip strength tests. This result demonstrates in vivo efficacy, recording significantly higher transcriptional activation fold-changes compared to existing ultra-miniature Cas tools.

Significance and Outlook

This work is evaluated as having expanded the scope of in vivo gene therapy from single-gene supplementation to complex gene expression regulation. It provides a direct clue for developing treatments for ultra-large gene deficiency diseases, which were difficult to package in existing carriers due to their massive size, as well as chronic metabolic diseases that require the simultaneous regulation of multiple signaling molecules.

The challenges to be addressed for commercialization and clinical application are also clear. The risk of off-target activation, in which unintended genes are stimulated outside the target site, must be rigorously controlled through whole-transcriptome analysis. It is also essential to conduct detailed verification procedures in non-clinical large animal models, such as primates, regarding potential immune responses or liver toxicity that may occur during long-term expression. Subsequent optimization studies are expected to continue, enhancing precision by combining tissue-specific promoters to ensure selective action only on target cells.