Engineering cell signaling using tunable CRISPR-Cpf1-based transcription factors.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Gene Regulation

Background:

• The catalytically dead Cpf1 endonuclease (dAsCpf1) from Acidaminococcus sp. BV3L6 is known for transcriptional repression in bacteria and plants.
• Its potential as a transcriptional regulator or signal conductor in human cells remains largely unexplored.

Purpose of the Study:

• To investigate the repurposing of the dAsCpf1 system in human cells for gene transcription regulation.
• To engineer programmable, ligand-controlled dAsCpf1 systems for creating artificial signaling pathways.

Main Methods:

• Repurposing the dAsCpf1 system for gene activation and repression in human cells.
• Constructing ligand-controlled systems by coupling crRNAs with riboswitches or fusing dAsCpf1 with G protein-coupled receptors.
• Regulating endogenous gene transcription in response to various ligands.

Main Results:

• Demonstrated the functionality of dAsCpf1 for both gene activation and repression in human cells.
• Developed programmable systems responsive to diverse ligands by integrating riboswitches or GPCRs.
• Observed signal amplification in systems processing multiple crRNAs from a single transcript.

Conclusions:

• The dAsCpf1 system offers a robust and efficient platform for engineering customized cell signaling circuits in human cells.
• This approach enables the construction of artificial signaling pathways with rewired cellular input-output behaviors.
• Programmable ligand-controlled dAsCpf1 systems provide a versatile tool for synthetic biology applications.