Guide RNA Lock Nucleic Acid Regulation
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Solution Overview
Problem
Current methods lack rapid, reversible, and temporal control over CRISPR-based technologies, particularly for inhibiting the activity of RNA-guided endonucleases like Cas9, which poses challenges in regulating genome editing and preventing malevolent use of gene drives.
Innovation Solution
The development of compositions and methods involving guide RNAs with regulatory domains that can be inactivated by lock nucleic acids and activated by trigger nucleic acids, allowing for conditional regulation of CRISPR-Cas systems by forming complexes with Cas enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas system is activated for genome editing, then gene editing capability is improved, but loss of temporal control and potential malevolent use increases
Solution Approach 1:
The guide RNA is designed with a regulatory domain that is pre-bound by a lock nucleic acid in an inactive state. This preliminary inactivation allows the system to be prepared and delivered without risking unintended genomic modifications, with activation occurring only after a specific trigger is introduced at the desired time point.
Solution Approach 2:
The system transitions from a static inactive state (locked by lock nucleic acid) to a dynamic active state upon trigger introduction. The regulatory domain of the guide RNA can dynamically switch between bound and unbound states, enabling temporal control of Cas9 activity according to the desired editing timeline.
2Productivity
If CRISPR-Cas system is activated for gene drive, then genetic modification efficiency is improved, but risk of malevolent use and unintended consequences increases
Solution Approach 1:
A trigger nucleic acid serves as an intermediary between the inactive CRISPR system and its activation. This intermediary component must be introduced separately to activate the system, creating an additional layer of control that prevents accidental or unauthorized activation while maintaining high efficiency when properly activated.
Solution Approach 2:
The regulatory domain is specifically designed with a toehold sequence that has high affinity for the lock nucleic acid. This localized high-affinity binding region ensures that the guide RNA remains tightly locked in the absence of trigger, preventing premature activation and reducing risks of malevolent use while allowing efficient activation when trigger is present.
3Reliability
If lock nucleic acid binds to guide RNA regulatory domain, then CRISPR-Cas activity is inhibited, but system complexity increases
Solution Approach 1:
The regulatory domain is merged into the guide RNA structure itself, with the toehold sequence being an integral part of the guide RNA. This merging eliminates the need for separate regulatory components, reducing overall system complexity while maintaining reliable inhibition through the lock nucleic acid binding to the integrated regulatory domain.
4Speed
If trigger nucleic acid displaces lock nucleic acid, then CRISPR-Cas activation speed is improved, but control precision may be reduced
Solution Approach 1:
The system employs a two-stage periodic activation process: first, the trigger nucleic acid binds to the toehold sequence; second, this binding initiates strand displacement that releases the lock nucleic acid and activates Cas9. This periodic action ensures controlled activation speed while maintaining precision through the sequential nature of the displacement mechanism.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables precise and reversible control over CRISPR-Cas activities, providing a mechanism to inhibit or activate genome editing as needed, addressing the limitations of existing technologies in terms of dosage and temporal control.
Implementation Method 1
lock nucleic acid different from the guide RNA; and displacing the lock nuclei acid from the regulatory domain
Implementation Method 2
displacing the lock nuclei acid from the regulatory domain by a trigger nucleic acid, thereby activating the guide RNA
Implementation Method 3
the activated guide RNA forms a complex with a Cas enzyme
Data Source
AI summary
Compositions and methods for conditionally regulating activities of CRISPR-Cas systems. In some embodiments, the methods comprise providing an inactive guide RNA comprising a regulatory domain bound by a lock nucleic acid different from the guide RNA; and displacing the lock nuclei acid from the regulatory domain by a trigger nucleic acid, thereby activating the guide RNA, wherein the activated guide RNA forms a complex with a Cas enzyme.