CRISPR/Cas System With Tetracycline Operator For Gene Editing Control
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Solution Overview
Problem
There is a lack of efficient and effective materials and methods for controlling the expression of CRISPR-related genes after they are delivered to cells, leading to potential detrimental effects.
Innovation Solution
The development of a CRISPR/Cas system that includes a nuclease segment, a guide RNA (gRNA) segment, and a promoter segment with a tetracycline operator sequence, allowing for controlled transcriptional and post-transcriptional regulation of Cas nuclease expression using a tetracycline repressor protein and short-hairpin RNA (shRNA).
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-related genes are continuously expressed after delivery to cells, then gene editing function is maintained, but detrimental effects occur and safety is compromised
Solution Approach 1:
The patent implements dynamic control of CRISPR gene expression through inducible promoter systems (e.g., tetracycline-responsive promoters) that allow the expression levels of Cas nuclease and gRNA to be adjusted in real-time based on therapeutic needs, rather than continuous static expression
Solution Approach 2:
The patent employs periodic or transient expression strategies where CRISPR components are expressed only during specific time windows (e.g., during AAV packaging or at specific therapeutic intervals) using inducible promoters that can be activated or deactivated as needed, preventing continuous expression-related toxicity
2Productivity
If CRISPR components are expressed during AAV packaging, then vector production is achieved, but mutagenesis at SIN sites occurs
Solution Approach 1:
The patent separates the CRISPR expression control from the AAV packaging process by using inducible promoters that can be selectively activated only after packaging is complete, or by using separate expression cassettes that are independently regulated, thereby extracting the harmful mutagenesis effect from the packaging process
Solution Approach 2:
The patent performs preliminary packaging of AAV vectors without active CRISPR expression, then activates CRISPR expression only after packaging is complete through inducible promoter systems, preventing mutagenesis during the critical packaging phase while still achieving gene editing function
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 enables precise control over CRISPR activity, reducing mutagenesis at specific sites in recombinant AAV vectors during packaging and ensuring safe and efficient gene editing.
Implementation Method 1
a promoter segment comprising a nucleotide sequence that encodes a first promoter comprising one or more tetracycline operator sequence, wherein the gRNA segment is operably linked to the promoter segment
Implementation Method 2
controlling post-transcriptional expression of Cas nuclease using a short-hairpin RNA (shRNA)
Data Source
AI summary
The present application provides materials and methods for controlling gene editing. The present application also provides materials and methods for controlling transcriptional expression of guide RNAs and/or post-transcriptional expression of Cas nuclease.