Self-inactivating Endonuclease Nucleic Acids for Transient Genome Editing
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Solution Overview
Problem
Current genome editing technologies face challenges in controlling the expression and activity of sequence-specific endonucleases, leading to potential off-target effects and inflammatory responses due to persistent enzyme activity.
Innovation Solution
A functional nucleic acid is designed to encode a sequence-specific endonuclease with endonuclease-recognized sequences that allow the nucleic acid to be cleaved by the expressed enzyme, thereby self-inactivating and preventing further expression, ensuring transient endonuclease activity and reducing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If sequence-specific endonucleases are expressed continuously for genome editing, then editing efficiency is improved, but off-target effects and inflammatory responses increase
Solution Approach 1:
The patent applies dynamics by making the endonuclease expression transient rather than continuous. The self-inactivating nucleic acid design allows the endonuclease to be expressed only temporarily until it cleaves its own encoding sequence, dynamically adjusting enzyme levels to achieve sufficient editing activity while preventing persistent off-target effects and inflammatory responses
Solution Approach 2:
The patent implements feedback control through the self-inactivating mechanism. The endonuclease product feeds back to cleave its own encoding sequence, creating a negative feedback loop that automatically terminates expression. This feedback mechanism ensures adequate enzyme levels for genome editing while preventing excessive accumulation that would cause harmful off-target effects
2Reliability
If the endonuclease encoding sequence is protected from cleavage, then enzyme expression is maintained, but loss of cellular control over endonuclease activity occurs
Solution Approach 1:
The patent applies self-service through the self-inactivating mechanism where the endonuclease automatically cleaves its own encoding sequence. This self-service approach allows the enzyme to maintain its own expression levels initially, then autonomously terminate its own production, providing cellular control without requiring external regulatory mechanisms
Solution Approach 2:
The patent implements preliminary anti-action by designing the endonuclease to recognize and cleave its own encoding sequence. This built-in anti-action is prepared in advance through the specific sequence design, allowing the cell to automatically control endonuclease activity levels and prevent uncontrolled expression before harmful effects occur
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
This approach provides controlled, transient genome editing activity with reduced off-target cleavage and inflammatory responses, enhancing the safety and specificity of genome editing processes.
Implementation Method 1
Sequence-specific endonucleases are DNA-cleaving enzymes that recognize a target nucleic acid by binding to a target sequence in the target nucleic acid creating double-stranded breaks (DSBs) or single-stranded nicks in the target nucleic acid upon binding
Data Source
AI summary
Disclosed herein is a functional nucleic acid that includes i) one or more coding nucleotide sequences encoding a genome editing endonuclease; ii) regulatory sequences operably linked to the one or more coding nucleotide sequences; and iii) one or more genome editing endonuclease-recognized sequences, wherein the functional nucleic acid is configured to express the endonuclease in a host cell and thereby provide a cellular endonuclease activity in a sequence-specific manner in the host cell, and wherein cleavage of the functional nucleic acid by the endonuclease inactivates the cellular endonuclease activity. Methods of using the present functional nucleic acid, and systems and kits that find use in performing the same are also provided.