Compact engineered human mechanosensitive transactivation modules enable potent and versatile synthetic...

Area of Science:

• Synthetic biology
• Molecular biology
• Gene regulation

Background:

• Programmable DNA-binding platforms combined with engineered transactivation domains (TADs) are crucial for synthetic transcriptional control.
• Current CRISPR-based transcriptional activation (CRISPRa) technologies often utilize large or poorly tolerated TADs, limiting their applications.

Purpose of the Study:

• To define and optimize minimal TADs from human mechanosensitive transcription factors.
• To develop potent and compact transactivation modules and a CRISPRa platform for enhanced synthetic transcriptional control.

Main Methods:

• Engineered minimal TADs from human mechanosensitive transcription factors.
• Constructed multipartite transactivation modules (MSN, NMS, eN3x9) and the CRISPR-DREAM platform.
• Tested CRISPR-DREAM specificity, robustness, and transcriptional stimulation across cell types and regulatory loci.
• Assessed portability of MSN and NMS across different CRISPR systems, TAL effectors, and zinc finger proteins.
• Demonstrated efficacy in reprogramming human fibroblasts and in primary human cell types.
• Developed dual and all-in-one CRISPRa adeno-associated virus (AAV) systems.

Main Results:

• Developed potent and compact transactivation modules (MSN, NMS, eN3x9) and the CRISPR-DREAM platform.
• CRISPR-DREAM showed specificity and robustness across mammalian cell types, efficiently activating diverse regulatory loci.
• MSN and NMS modules demonstrated portability across various CRISPR systems, TAL effectors, and zinc finger proteins.
• Successfully reprogrammed human fibroblasts into induced pluripotent stem cells using dCas9-NMS.
• Mechanosensitive transcription factor TADs proved efficacious and well-tolerated in primary human cells.
• Engineered compact CRISPRa AAV systems for dual and all-in-one applications.

Conclusions:

• Compact human TADs derived from mechanosensitive transcription factors offer potent and well-tolerated synthetic transcriptional control.
• The developed transactivation modules and CRISPR-DREAM platform are versatile and applicable across different systems.
• These advancements facilitate improved CRISPRa technologies for biomedical applications, including cell reprogramming and gene therapy.