CRISPR Cascade Diagnostics with Aptamer-Controlled Reporter Enzymes
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Solution Overview
Problem
Existing CRISPR-based diagnostics face limitations in sensitivity and require additional steps such as nucleic acid amplification or separation, which can complicate the workflow and reduce efficiency.
Innovation Solution
A novel reaction mixture and method utilizing a CRISPR enzyme, guide RNA, and nucleic acid aptamer to modulate the activity of a reporter enzyme, allowing for enhanced sensitivity and detection of target nucleic acids without the need for separation steps or nucleic acid amplification, through enzymatic cascade reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-based diagnostics use traditional methods with nucleic acid amplification and separation steps, then detection capability is achieved, but workflow complexity increases and efficiency decreases
Solution Approach 1:
The patent combines multiple diagnostic steps (detection, amplification, separation) into a single integrated CRISPR reaction mixture. The guide RNA and CRISPR enzyme work together in one pot to perform target recognition, signal amplification, and detection without requiring separate amplification or separation steps, thus reducing workflow complexity while maintaining detection capability.
Solution Approach 2:
The CRISPR reaction mixture is designed to perform multiple functions simultaneously: target sequence recognition, signal amplification through enzymatic cascade reactions, and direct detection. This multi-functional system eliminates the need for specialized reagents and equipment for separate amplification and separation steps.
2Reliability
If CRISPR-based diagnostics use traditional methods with additional processing steps, then detection is performed, but time consumption increases
Solution Approach 1:
The reaction mixture is pre-configured with all necessary components (CRISPR enzyme, guide RNA, substrates) before sample addition. This preliminary preparation allows the detection reaction to proceed immediately without requiring time-consuming setup of separate amplification and separation systems, reducing overall time consumption while maintaining detection accuracy.
3Productivity
If CRISPR-based diagnostics use simplified workflows without amplification, then efficiency improves, but sensitivity decreases
Solution Approach 1:
The patent introduces an intermediary enzymatic cascade reaction system that amplifies the CRISPR recognition signal. The CRISPR enzyme activates downstream enzymes (such as polymerases or nucleases) that produce detectable signals through cascade reactions, achieving signal amplification without requiring traditional nucleic acid amplification steps, thus maintaining both efficiency and sensitivity.
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
The approach achieves improved sensitivity and specificity in detecting target nucleic acids, with a limit of detection as low as ~10 fM, and eliminates the need for additional steps, providing a more streamlined workflow.
Implementation Method 1
a CRISPR enzyme (also referred to herein as a CRISPR-Cas enzyme or CRISPR nuclease) with nuclease activity
Implementation Method 2
when region of the gRNA is hybridized to a pre-determined target polynucleotide acid sequence
Implementation Method 3
a nucleic acid aptamer capable of binding to the second enzyme. The second enzyme and the aptamer may be in a complex comprising the second enzyme and the aptamer
Data Source
AI summary
The present disclosure relates to enzymatic cascade reactions for achieving better sensitivity in detecting a target nucleic acid sequence. Several aspects of the disclosure relate to a reaction master mix comprising four major components: a CRISPR enzyme, a guide RNA (gRNA), an aptamer (e.g., an inhibiting aptamer or an activating aptamer) and a “signaling” (i.e., reporter) enzyme. The aptamer interacts with the signaling enzyme and forms a complex, resulting in, e.g., inhibited signaling enzyme activity. When a target is present in the reaction mix, the Cas/guideRNA system becomes activated and preferentially collaterally cleave(s) all nucleic acids in the solution, including the aptamer. Once aptamer gets cleaved, the signaling enzyme is free to produce a signal. In the presence of a substrate, such signaling enzyme activity produces a robust signal that significantly improves the limit-of-detection of other techniques in the art.