spostrzeżenie - Synthetic Biology - # Protein-Protein Proximity Triggered Genome Editing

A Molecular Proximity Sensor for Programmable Genome Editing via Engineered Guide RNA

Q: How could the P3 editing strategy be further expanded to enable multiplexed, signal-specific genome editing within the same cell

To enable multiplexed, signal-specific genome editing within the same cell using the P3 editing strategy, several key expansions can be considered: Orthogonal Protein-RNA Pairs: Identifying and utilizing additional orthogonal protein-RNA pairs beyond MS2-MCP and BoxB-LambdaN can allow for the development of multiple independent P3 editing systems within the same cell. By incorporating different protein-RNA interactions, each specific to a different signal or event, the cell can respond to a variety of stimuli with distinct genome editing outcomes. Optimized Dimerization Sequences: Further optimization of the dimerization sequences within the crRNA and petracrRNA can enhance the efficiency and specificity of P3 editing. By fine-tuning the base composition, length, and positioning of the dimerization regions, the system can be tailored to respond more robustly to specific protein-protein interactions. Multiplexed Sensor Integration: Integrating multiple P3 editing systems with different protein-protein interactions into a single cell can create a platform for multiplexed sensing and editing. Each P3 editing system can be linked to a unique protein interaction, allowing the cell to respond to a combination of signals and trigger distinct genome editing events simultaneously. Signal Amplification Strategies: Implementing signal amplification strategies within the P3 editing system can enhance the sensitivity and responsiveness to low-abundance signals. By incorporating amplification mechanisms, such as cascades of protein interactions or enzymatic reactions, the system can amplify weak signals to achieve a more robust genome editing response. By incorporating these expansions, the P3 editing strategy can be extended to enable sophisticated, multiplexed, and signal-specific genome editing within the same cell, offering a versatile platform for precise control of cellular functions based on diverse molecular signals.

Q: What other types of molecular events, beyond protein-protein interactions, could potentially be transduced into genome editing outcomes using the P3 editing approach

The P3 editing approach, which transduces protein-protein interactions into genome editing outcomes, can be adapted to convert various other molecular events into precise genome editing responses. Some potential types of molecular events beyond protein-protein interactions that could be transduced using the P3 editing approach include: Small Molecule Induced Interactions: Leveraging small molecule-induced protein-protein interactions, such as rapamycin-induced dimerization or ABA-induced interactions, can be integrated into the P3 editing system. By linking small molecule-responsive protein pairs to the formation of active gRNA complexes, specific small molecule signals can trigger targeted genome editing events within the cell. Post-Translational Modifications: Incorporating post-translational modifications that induce protein-protein interactions, such as phosphorylation or ubiquitination, into the P3 editing system can enable the conversion of these modifications into genome editing outcomes. By designing sensor components that respond to specific modifications, the system can translate dynamic cellular signaling events into precise genetic modifications. RNA-Protein Interactions: Expanding the P3 editing approach to include RNA-protein interactions, where specific RNA molecules bind to target proteins, can enable the detection of RNA-based signals and their conversion into genome editing responses. By engineering RNA aptamers that interact with specific proteins, the system can respond to RNA expression patterns and induce tailored genome edits based on RNA signals. By broadening the scope of molecular events that can be transduced into genome editing outcomes using the P3 editing approach, the system can offer a versatile platform for controlling cellular functions in response to diverse signaling cues.

Główne pojęcia

A molecular proximity sensor based on an engineered, dual-component guide RNA can link specific protein-protein interactions or chemically-induced dimerization to the activation of CRISPR-based genome editing tools, enabling programmable control of genome editing outcomes.

Streszczenie

The article presents a strategy called "P3 editing" (protein-protein proximity) that links protein-protein proximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. The key insights are:

The authors tested three strategies to split the prime editing guide RNA (pegRNA) into two parts, with the goal of requiring specific protein-protein interactions to restore the functional guide RNA. They found that splitting the pegRNA at the crRNA:tracrRNA junction was the most promising approach.
The authors then demonstrated that by replacing the crRNA:tracrRNA dimerization domain with RNA aptamers that bind specific protein domains (e.g. MS2-MCP, BoxB-LambdaN), they could use known protein-protein interactions or chemically-induced dimerization to drive the formation of the active guide RNA complex and induce genome editing.
They showed that this P3 editing strategy could be coupled to both base editing and prime editing tools, providing a way to link a variety of protein-based signals to specific genome editing outcomes.
The authors also explored combining the P3 editing approach with an ADAR-based RNA sensor system, demonstrating the potential to control genome editing based on the expression of specific target RNAs.
The authors characterized the efficiency-specificity tradeoff in their P3 editing designs, and discussed strategies to further optimize the system for multiplex applications.

Overall, the P3 editing approach provides a versatile way to program genome editing outcomes based on diverse molecular events occurring within living cells.

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biorxiv.org

Statystyki

One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs.
Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA.
The CRISPR system for immune response in bacteria has been repurposed as a programmable genome editing method.
Prime editing extends the programmability of CRISPR by using a prime editing guide RNA (pegRNA) that programs both the target locus and editing outcome.

Cytaty

"One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs."
"Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA."
"The CRISPR system for immune response in bacteria has been repurposed as a programmable genome editing method."
"Prime editing extends the programmability of CRISPR by using a prime editing guide RNA (pegRNA) that programs both the target locus and editing outcome."

Kluczowe wnioski z

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

by Choi,J., Che... o www.biorxiv.org 08-14-2023

https://www.biorxiv.org/content/10.1101/2023.08.14.553235v2

Głębsze pytania

How could the P3 editing strategy be further expanded to enable multiplexed, signal-specific genome editing within the same cell

To enable multiplexed, signal-specific genome editing within the same cell using the P3 editing strategy, several key expansions can be considered:

Orthogonal Protein-RNA Pairs: Identifying and utilizing additional orthogonal protein-RNA pairs beyond MS2-MCP and BoxB-LambdaN can allow for the development of multiple independent P3 editing systems within the same cell. By incorporating different protein-RNA interactions, each specific to a different signal or event, the cell can respond to a variety of stimuli with distinct genome editing outcomes.

Optimized Dimerization Sequences: Further optimization of the dimerization sequences within the crRNA and petracrRNA can enhance the efficiency and specificity of P3 editing. By fine-tuning the base composition, length, and positioning of the dimerization regions, the system can be tailored to respond more robustly to specific protein-protein interactions.

Multiplexed Sensor Integration: Integrating multiple P3 editing systems with different protein-protein interactions into a single cell can create a platform for multiplexed sensing and editing. Each P3 editing system can be linked to a unique protein interaction, allowing the cell to respond to a combination of signals and trigger distinct genome editing events simultaneously.

Signal Amplification Strategies: Implementing signal amplification strategies within the P3 editing system can enhance the sensitivity and responsiveness to low-abundance signals. By incorporating amplification mechanisms, such as cascades of protein interactions or enzymatic reactions, the system can amplify weak signals to achieve a more robust genome editing response.

By incorporating these expansions, the P3 editing strategy can be extended to enable sophisticated, multiplexed, and signal-specific genome editing within the same cell, offering a versatile platform for precise control of cellular functions based on diverse molecular signals.

What other types of molecular events, beyond protein-protein interactions, could potentially be transduced into genome editing outcomes using the P3 editing approach

The P3 editing approach, which transduces protein-protein interactions into genome editing outcomes, can be adapted to convert various other molecular events into precise genome editing responses. Some potential types of molecular events beyond protein-protein interactions that could be transduced using the P3 editing approach include:

Small Molecule Induced Interactions: Leveraging small molecule-induced protein-protein interactions, such as rapamycin-induced dimerization or ABA-induced interactions, can be integrated into the P3 editing system. By linking small molecule-responsive protein pairs to the formation of active gRNA complexes, specific small molecule signals can trigger targeted genome editing events within the cell.

Post-Translational Modifications: Incorporating post-translational modifications that induce protein-protein interactions, such as phosphorylation or ubiquitination, into the P3 editing system can enable the conversion of these modifications into genome editing outcomes. By designing sensor components that respond to specific modifications, the system can translate dynamic cellular signaling events into precise genetic modifications.

RNA-Protein Interactions: Expanding the P3 editing approach to include RNA-protein interactions, where specific RNA molecules bind to target proteins, can enable the detection of RNA-based signals and their conversion into genome editing responses. By engineering RNA aptamers that interact with specific proteins, the system can respond to RNA expression patterns and induce tailored genome edits based on RNA signals.

By broadening the scope of molecular events that can be transduced into genome editing outcomes using the P3 editing approach, the system can offer a versatile platform for controlling cellular functions in response to diverse signaling cues.

What are the potential applications of a system that can precisely control genome editing based on specific RNA expression patterns within a cell

A system that can precisely control genome editing based on specific RNA expression patterns within a cell holds significant potential for various applications in synthetic biology and biotechnology. Some key potential applications include:

RNA-Based Disease Detection and Treatment: The ability to link RNA expression patterns to targeted genome editing events can enable the development of diagnostic tools for detecting specific RNA signatures associated with diseases. By programming the system to respond to disease-related RNA molecules, it can trigger therapeutic genome edits to correct genetic mutations or modulate gene expression, offering a personalized approach to disease treatment.

Cellular Reprogramming and Differentiation: By integrating RNA expression-based control of genome editing, the system can be used to guide cellular reprogramming and differentiation processes. Specific RNA expression patterns associated with stem cell pluripotency or differentiation stages can be used to direct precise genome edits that drive cell fate transitions, offering new avenues for regenerative medicine and tissue engineering.

Dynamic Cellular Response Systems: The system can be employed to create dynamic cellular response systems that adapt to changing environmental cues or internal signals. By linking RNA expression patterns to genome editing outcomes, cells can be engineered to respond to specific stimuli with tailored genetic modifications, allowing for on-demand control of cellular functions in response to varying conditions.

Biological Circuit Design: Integrating RNA-based control of genome editing into synthetic biological circuits can enable the construction of complex genetic networks with feedback loops and signal processing capabilities. By incorporating RNA sensors that drive specific genome edits, the system can be used to design sophisticated molecular circuits that process information and execute precise genetic programs within living cells.

Overall, a system that can control genome editing based on specific RNA expression patterns offers a versatile tool for manipulating cellular functions with high precision, opening up diverse opportunities in research, therapeutics, and biotechnology.