A molecular proximity sensor based on an engineered, dual-component guide RNA.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genome Engineering

Background:

• Synthetic biology aims to create programmable molecular circuits in living cells.
• Genome editing tools like CRISPR-Cas9 are crucial for these circuits but programming their activation is challenging.
• Controlling genome editing via molecular events like protein interactions is a key unmet need.

Purpose of the Study:

• To develop a novel strategy for programming CRISPR-Cas9 genome editing using protein-protein interactions.
• To demonstrate the activation of prime editing and base editing through engineered proximity-dependent guide RNAs.
• To explore the integration of RNA sensing pathways for RNA-inducible genome editing.

Main Methods:

• Engineered a dual-component guide RNA for CRISPR-Cas9 systems.
• Linked protein-protein interactions and chemically induced dimerization to guide RNA formation.
• Tested the P3 editing strategy in human cells for prime editing and base editing.
• Integrated ADAR-based RNA sensors to trigger genome edits.

Main Results:

• Demonstrated that protein proximity can activate CRISPR-Cas9 prime editing and base editing.
• Showcased the use of various known protein-protein interactions and chemical inducers.
• Successfully linked RNA sensing pathways to specific genome editing events.
• Enhanced the controllability of CRISPR-based genome editing in synthetic circuits.

Conclusions:

• P3 editing provides a robust method to control genome editing with molecular inputs.
• This strategy significantly advances the development of sophisticated synthetic molecular circuits.
• Enables precise and programmable genome modifications within living cells for diverse applications.