Self-Inactivating CRISPR Nuclease for Stable Genome Editing
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Solution Overview
Problem
Current genome editing systems, particularly CRISPR-based single-base editing systems, face challenges with instability and off-targeting, leading to potential risks of unintended mutations and continuous editing of already modified sequences.
Innovation Solution
Incorporating a gRNA that targets the coding sequence of the CRISPR nuclease within the genome editing system, causing an inactivating mutation that terminates the CRISPR nuclease's activity, thereby preventing further editing and enhancing specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If a CRISPR-based single base editing system is used to achieve precise base substitution, then manufacturing precision is improved, but reliability deteriorates due to potential off-targeting and continuous editing of already modified sequences
Solution Approach 1:
The patent applies preliminary anti-action by designing a suicide gRNA that targets and inactivates the CRISPR nuclease gene before the editing system can cause harmful off-target effects or continuous editing. The suicide gRNA is expressed alongside the editing gRNA, and after a delay period allowing the editing to complete, the suicide gRNA takes effect to destroy the nuclease gene, preventing any subsequent unwanted editing events.
Solution Approach 2:
The patent uses a delay element as an intermediary mechanism between the editing gRNA and the suicide gRNA. This delay element controls the timing of suicide gRNA expression, ensuring that the CRISPR nuclease remains active long enough to perform the desired base substitution but is subsequently inactivated to prevent off-target effects and continuous editing.
2Productivity
If the CRISPR nuclease remains active continuously to ensure complete editing, then productivity is improved, but object-generated harmful factors increase due to off-target mutations
Solution Approach 1:
The patent implements periodic action by dividing the CRISPR system operation into distinct phases: first, the editing gRNA directs the nuclease to perform the desired base substitution; then, after a controlled delay, the suicide gRNA is expressed to inactivate the nuclease. This temporal separation ensures that the nuclease is active only when needed for productive editing and is subsequently deactivated to prevent harmful off-target mutations.
3Reliability
If a suicide gRNA targeting the CRISPR nuclease is introduced to prevent continuous editing, then reliability is improved, but device complexity increases
Solution Approach 1:
The patent applies universality by designing the suicide gRNA to recognize and target the CRISPR nuclease gene itself, making the system self-regulating. The same CRISPR machinery that performs the beneficial editing is also used by the suicide gRNA to inactivate itself, eliminating the need for separate degradation mechanisms or complex control systems while ensuring reliable mutation stability.
Data Source
AI summary
Provided are an improved genome editing system and a method that has high specificity, which are capable of obtaining stable mutation types. The system includes an expression construct including a coding sequence of a gRNA targeting at least one genomic target sequence; an expression construct including a coding sequence of a CRISPR nuclease; and an expression construct including a coding sequence for a gRNA targeting a target sequence within the coding sequence of the CRISPR nuclease. Upon introduction into the cell, the gRNA targeting the at least one genomic target sequence directs the CRISPR nuclease to the at least one genomic target sequence and results in one or more mutations in the genomic target sequence, and the gRNA targeting a target sequence within the coding sequence of the CRISPR nuclease directs the CRISPR nuclease to the target sequence within the coding sequence of the CRISPR nuclease and results in an inactivating mutation of the CRISPR nuclease.