Tiny CRISPR molecular switch tool opens a new world

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In recent years, the CRISPR-Cas9 system, which has been applied in various living systems to cut a target gene and induce reprogrammed gene editing, has gained considerable attention.¹ Building on these CRISPR systems, a new technology is being developed to improve the current systems and render them safe for in vivo application. This technology allows for the control of gene expression regulation without altering the DNA sequence. Commonly referred to as inactivating CRISPR-Cas9-based transcription activator (CRISPRa [CRISPR activator]) or inactivating CRISPR-Cas9-based transcription interference (CRISPRi [CRISPR interference]) systems,^2,3 these advancements are designed to bind to sequences near the transcription start site of the target gene. This binding results in either amplification or repression of the expression of the target gene depending on the system used.

In particular, optimization studies are underway to develop CRISPR-Cas systems based on fewer immunogenic delivery vehicles, such as adeno-associated virus (AAV), for gene therapy applications involving effective gene expression regulation systems in humans.⁴ Although AAV-mediated delivery has many advantages for human applications, gene size restrictions for packaging foreign genes have been pointed out as a limitation in gene therapy development using CRISPR-Cas systems. Consequently, ultra-small CRISPR-Cas systems are gaining attention for effective AAV viral delivery^5,6,7 (Figure 1). In a recent paper published in Molecular Therapy, Wang et al. showed the potential for developing AAV-system-based gene therapies using an innovative and efficient inactive CRISPR-Cas12f system (called dUn1Cas12f-3R).⁸ In their study, the authors engineered the protein component of the recently discovered CRISPR-Cas12f system to develop a system that can effectively replace conventional CRISPR-Cas9-based transcriptional amplification or repression systems. The authors’ highly efficient, inactivated dUn1Cas12f (Sso7d-dUn1Cas12f1-3R)-based transcriptional activator (Sso7d-dUn1Cas12f1-3R-VPR [VP64-P65-RTA], SminiCRa) or repressor (Sso7d-dUn1Cas12f1-3R-KRAB, SminiCRi) system enables high-accuracy gene expression regulation by targeting thymine-rich gene targets. This capability allows for the simultaneous targeting of multiple genes in a cell and gene expression regulation across various human-derived cell lines, which is not achievable with conventional CRISPR-Cas9 systems. The authors demonstrated that the dUn1Cas12f1-3R-KRAB (transcriptional repressor, miniCRi) system, a compact-size system with efficient DNA-binding properties, was delivered into human-derived cells by AAV and effectively suppressed the expression of mutated genes related to genetic diseases. These findings highlight the ability of the tiny molecular switch to block the expression of dysfunctional genes. These promising results indicate that the effective delivery of the SminiCRa or SminiCRi system through systemic or local injection in humans has the potential to be developed as a gene therapy for various diseases caused by genetic mutations.

Figure 1.

Limitations on the size of genes that can be loaded to AAV viruses for human delivery

Advanced forms of CRISPR gene expression regulatory tools, such as ultra-small, enhanced versions of CRISPR, can be effectively delivered to humans via AAV for gene therapy purposes. dUnCas12f-3R: inactive Cas12f (D143R, T147R, T203R) from uncultured archaeon; VP64, p65, and RTA: transcriptional activator domains; KRAB: transcriptional repression domain; Sso7d: a nonspecific dsDNA-binding protein. Images were created with BioRender.com.

The recently discovered Cas12f system has shown promise for effective gene editing when introduced in vivo using AAVs because of its highly accurate recognition of target DNA as well as the small gene size for AAV packaging.^5,6,7 In previous studies, a system that can regulate intracellular gene expression without cutting DNA, through the connection of activation factors such as VPR, was developed based on an inactive CRISPR-Cas12f system (dCRISPR-Cas12f). However, its efficiency was not high for various genes, making it difficult to apply to actual living systems.⁷ In this study, Wang et al. introduced amino acid substitutions into the CRISPR-Cas12f protein (dUn1CRISPR-Cas12f1-3R, D143R, T147R, and T203R) to enhance its interaction with target DNA.⁸ Additionally, nonspecific DNA-binding modules, such as Sso7d, were connected (Sso7d-dUn1CRISPR-Cas12f1-3R) to further enhance their functional activity on target DNA. The inactive dCas12f system (dUn1Cas12f) with this enhanced target DNA binding resulted in significant gene expression enhancement or repression activity against a variety of genes in human-derived cell lines depending on the ligation of the VPR domain (Sso7d-dUn1CRISPR-Cas12f1-3R-VPR, SminiCRa) or the KRAB domain (Sso7d-dUn1CRISPR-Cas12f1-3R-KRAB, SminiCRi). The gene expression modulation efficiency was stronger than that of the dCRISPR-Cas12f or dCasMINI versions previously developed with the inactivated dCRISPR-Cas12f system and comparable to that of the dCas9-VPR (CRISPRa) or dCas9-KRAB (CRISPRi) systems, which are already well known for their ability to recognize target DNA and allow for robust DNA expression modulation functionality. Surprisingly, this gene expression modulation activity was able to operate simultaneously on multiple genes in the cell. Moreover, the authors demonstrated that this enhanced dUn1Cas12f1-3R-based gene suppression system (miniCRi) could be applied to AAVs to target the SERPINA1 gene and work in human-derived Huh-7 cells. This indicates that it could lead to the development of gene therapies using enhancement and suppression of targeted gene expression in humans in the future.

In this study, the authors demonstrated the effective gene expression amplification and suppression efficiency of the CRISPR-Cas12f system at the basic research level in human-derived cells. However, for clinical translational research, confirming the efficacy and safety of this gene expression regulation system through effective application to various animal experiments, including small animals, is necessary in the near future.

Various gene therapy attempts have been carried out in humans to date, and the CRISPR system has enabled the targeting, induction of mutations, and expression control of desired genes through easy reprogramming, thus heralding the era of fundamental gene therapy at the molecular level.⁹ To apply these CRISPR-based gene expression regulation systems to genes in the human body, effective delivery to living tissues and safe gene targeting are the main issues.^4,10 The authors’ work presented here validates a safe and effective gene expression induction technology based on ultrasmall molecular switch systems, thereby laying the foundation for future research on CRISPR-based gene therapy applications in humans. Being able to effectively control the expression of mutated genes in humans through single modifications at the molecular level using a miniaturized CRISPR switch system represents an important step toward the development of innovative gene therapies.