Establishing artificial gene connections through RNA displacement-assembly-controlled CRISPR/Cas9 function

Area of Science:

• Synthetic biology
• Molecular and cellular biology
• Genetic engineering

Background:

• Reprogramming genetic networks and signal pathways via synthetic circuits is crucial for biosystem manipulation.
• Building artificial genetic communications among endogenous RNA species is challenging due to sequence independence and structural diversity.

Purpose of the Study:

• To develop an RNA-based synthetic circuit for establishing regulatory linkages between endogenous gene expression in bacteria and mammalian cells.
• To demonstrate the effectiveness of this circuit for creating artificial connections between unrelated genes and controlling cellular phenotypes.

Main Methods:

• Utilized a displacement-assembly approach to modulate guide RNA activity for CRISPR/Cas9 function control.
• Engineered synthetic RNA circuits capable of linking endogenous RNA species.

Main Results:

• Successfully established regulatory linkages between endogenous gene expression in both Escherichia coli and mammalian cells.
• Demonstrated that exogenous and naturally occurring RNAs (small/microRNAs, long mRNAs) can control endogenous gene expression.
• Created an artificial signal pathway in mammalian cells to control cell apoptosis using the synthetic circuit.

Conclusions:

• The developed RNA-based synthetic circuit provides a general strategy for constructing artificial connections within genetic networks.
• This approach enables the reprogramming of cellular functions and alteration of phenotypes in mammalian cells.
• Offers a powerful tool for synthetic biology applications and understanding gene regulation.