Conditional CRISPR sgRNA Expression via Recombinase Switching
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR/Cas9 systems face challenges in achieving predictable and high cutting efficiency with high specificity, particularly in regulating guide RNA expression, and lack tight inducible systems for turning sgRNA expression on or off.
Innovation Solution
The development of an expression cassette with recombinase recognition sites that allows for conditional expression of sgRNA through recombinase-mediated recombination, enabling 'off-to-on' or 'on-to-off' switching by excising transcription disruption sequences or required elements within the promoter or sgRNA sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a constitutive promoter is used for sgRNA expression, then high expression levels are achieved, but tight inducible control (ON/OFF switching) is lost
Solution Approach 1:
The invention converts a static constitutive promoter system into a dynamic inducible system by incorporating recombinase recognition sites (loxP or FRT) that enable temporal control. The promoter activity can be switched ON or OFF by inducing recombinase expression, allowing dynamic regulation of sgRNA production while maintaining high expression levels when activated.
Solution Approach 2:
The invention introduces recombinase recognition sites as intermediary elements between the promoter and sgRNA sequence. These sites serve as molecular switches that, when bound by recombinase, alter promoter accessibility or activity, thereby mediating inducible control without directly modifying the promoter sequence itself.
2Ease of operation
If an imperfect Tet-inducible system is used, then some inducible control is achieved, but leakiness and lack of tight ON/OFF switching occur
Solution Approach 1:
The invention extracts the control mechanism from the imperfect Tet-inducible system and implements a novel recombinase-based switching system. By removing reliance on Tet repressor dynamics and instead using irreversible or reversible recombinase-mediated recombination at loxP/FRT sites, the system achieves tight ON/OFF switching without the leakiness inherent in Tet-based systems.
Solution Approach 2:
The invention uses multiple copies of recombinase recognition sites (typically two or more loxP or FRT sites) arranged in specific orientations to create robust switching control. This multi-site arrangement ensures complete and reliable promoter activation or deactivation, overcoming the partial control achieved by single-site or imperfect Tet-inducible systems.
3Duration of action of stationary object
If sgRNA expression is not tightly regulated, then continuous CRISPR activity occurs, but specificity and efficiency are reduced
Solution Approach 1:
The invention implements periodic rather than continuous CRISPR activity through inducible sgRNA expression. By controlling the timing of recombinase induction, the system activates sgRNA production only during desired time windows, allowing multiple cycles of ON/OFF switching that enhance specificity by limiting off-target effects while maintaining high cutting efficiency during active periods.
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 tighter regulation of sgRNA expression, overcoming issues of leakiness and improving the specificity and efficiency of CRISPR/Cas9 activity, allowing for precise control of target DNA modification.
Implementation Method 1
recombinase activated recombination at the pair of recombinase recognition sites is capable of excising said flanked sequence
Data Source
AI summary
An expression cassette for conditional expression of a single guide RNA (sgRNA) of a CRISPR/Cas9 system, the cassette includes a promoter, an sgRNA sequence, and a sequence flanked by at least a pair of recombinase recognition sites, wherein recombinase activated re-combination at the pair of recombinase recognition sites is capable of excising said flanked sequence, whereby either i) at least one of said recombinase recognition sites is located within the sgRNA sequence and said flanked sequence contains an transcription disruption sequence or ii) said flanked sequence is at least a part of the promoter or of the sgRNA sequence; methods of conditional expression of sgRNA and a reaction product of the conditional expression, i.e. sgRNA with a recombination site remnant.