Progress in Precise Gene Expression Control With CRISPR-Cas12f

An innovative CRISPR-Cas12f-based activator system may hold significant potential in the future of gene therapy and biological research.

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Researchers developed an optimized CWCas12f-VPR gene expression activation system aimed at addressing the limitations of the existing CRISPR activator (CRISPRa) systems, offering a significant advantage in the field of gene therapy. The innovative system, referred to as eCWCas12f-VPR, integrates the CRISPR-Cas module with activation domains and various combinations of linkers and nuclear localization signals,significantly enhancing both efficiency and specificity in gene expression regulation. The findings are published Gene Therapy.¹

The study highlights the limitations of traditional CRISPR-Cas effectors, including weak activity and unsuitable sizes for adeno-associated virus (AAV) loading.² In contrast, the CRISPR-Cas12f system is characterized by its small domains and homodimeric target DNA recognition, which leads to improved target specificity. The research team demonstrated that the eCWCas12f-VPR system could achieve "an average gene expression regulation efficiency several times higher than that of conventional CRISPRa."

One of the enhancements includes the introduction of 2 variants of guide RNAs (ge4.0 and ge4.1) and the engineering of Cas proteins based on the CWCas12f effector. Researchers conducted experiments using human-derived HEK293FT cells and observed that the ge4.1 guide RNA version yielded higher average fluorescence intensity, indicating better gene activation efficiency. The researchers then introduced amino acid mutations and combined them with the FUS-IDR domain, known for recruiting transcription initiation factors through phase separation.

The study reported that the optimized version, eCWCas12f-VPR, "demonstrated an efficiency comparable to that of previously reported gene expression activation (CRISPRa) systems based on Cas9 or Cas12a."

In addition, the researchers tested multiplexed gene activation with the eCWCas12f-VPR system. Using a vector that co-expresses the Csy4 domain with eCWCas12f-VPR, the data found that the system could simultaneously induce the activation of multiple target genes (CD2, CXCR4, and HBG) through the expression of multiple guide RNAs. The study reports that gene expression levels for CD2, CXCR4, and HBG were upregulated by approximately 6.41-, 1.05-, and 7.04-fold, respectively, compared with the non-targeting control.

In terms of specificity, the eCWCas12f-VPR system demonstrated a significant increase in the target specificity of various gene loci within human-derived cells (HBB, ASCL1, HBG, CD2), with "an average improvement of 26.06-fold" compared with the existing LbCas12a-VPR system. Researchers also performed RNA-seq analyses on eCWCas12f-VPR and LbCas12a-VPR constructs to evaluate the whole transcriptome-wide specificity, concluding that the eCWCas12f-VPR system showed greater specificity and fewer off-target effects than the LbCas12a-VPR system. Specifically, eCWCas12f-VPR-based activation resulted in gene expression changes in 312 genes, compared to 808 genes affected by LbCas12a-VPR.

The study further explores the potential of the eCWCas12f-VPR system for in vivo applications using AAV-mediated gene delivery. Researchers designed dual rAAV2 vectors for eCWCas12f-VPR and its corresponding sgRNA expression. When delivered through AAV vectors, the eCWCas12f-VPR system significantly increased HBG gene expression by 6.55-fold, demonstrating its potential for activating endogenous genes within human-derived HEK293FT cells.

The study's findings emphasize the potential of the eCWCas12f-VPR system as a powerful tool for gene therapy. It provides precise control over gene expression, offering potential applications in various biological and medical fields.

"The eCas12f-VPR system developed in this study provides a foundation for future applications in various biomedical fields, including molecular level-based screening, identification of gene functions, and disease modeling," the authors wrote. "Furthermore, this targeted and specific gene expression control technology will likely lay the groundwork for the development of gene therapies applicable to humans."

References

1. Oh Y, Gwon LW, Lee HK, et al. Highly efficient and specific regulation of gene expression using enhanced CRISPR-Cas12f system. Gene Ther. 2024;31(7-8):358-365. doi:10.1038/s41434-024-00458-w

2. Asmamaw Mengstie M. Viral vectors for the in vivo delivery of CRISPR components: advances and challenges. Front Bioeng Biotechnol. 2022;10:895713. Published May 12, 2022. doi:10.3389/fbioe.2022.895713