TRACR RNA-Dependent CRISPR Nuclease for Specific RNA Detection

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current CRISPR technologies for nucleic acid detection rely on non-specific nuclease activity, whereas the proposed method utilizes sequence-specific tracrRNA-dependent CRISPR nuclease activity for detecting RNAs, offering a distinct approach by generating non-canonical crRNAs from any RNA to link it to an active CRISPR nuclease for specific targeting.

Innovation Solution

The method involves contacting a sample with non-naturally occurring tracrRNA that specifically hybridizes with a sensed RNA, allowing it to bind or cleave a target nucleic acid in the presence of a tracrRNA-dependent CRISPR nuclease enzyme, enabling detection of the sensed RNA through binding or cleavage events.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If non-specific nuclease activity is used for nucleic acid detection, then detection capability is achieved, but specificity and off-targeting are compromised

Engineering Contradiction:

Improvedetection specificityVSAvoidoff-targeting

Core Design Contradiction:

Measurement precisionVSObject-affected harmful factors

Solution Approach 1:

The patent introduces a tracrRNA-dependent CRISPR nuclease system as an intermediary between the detected RNA target and the nuclease activity. The tracrRNA specifically hybridizes with the sensed RNA, forming a specific complex that recruits the CRISPR nuclease only to the correct target, thereby eliminating off-target effects while maintaining detection capability

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The invention applies local quality by making the nuclease activity conditional and location-specific. The CRISPR nuclease is engineered to be activated only when bound to the specific tracrRNA-sensed RNA complex, ensuring that cleavage activity occurs only at the correct target site and not elsewhere in the sample

Inventive Principle:

Principle #3Local quality

2Measurement precision

If sequence-specific tracrRNA-dependent CRISPR nuclease activity is used, then detection specificity is improved, but system complexity increases

Engineering Contradiction:

Improvedetection specificityVSAvoidsystem complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent employs a universal tracrRNA-dependent CRISPR nuclease system that can detect multiple different RNA targets by simply changing the tracrRNA sequence. The core nuclease enzyme remains the same, but its specificity is programmatically changed through different tracrRNA molecules, reducing overall system complexity while maintaining high detection specificity

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The invention changes the specificity parameter of the nuclease system by modifying the tracrRNA sequence rather than changing the nuclease itself. This allows the same nuclease enzyme to be reused for detecting different RNA targets, simplifying the system architecture while achieving sequence-specific detection

Inventive Principle:

Principle #35Parameter changes

3Adaptability or versatility

If reprogrammed tracrRNAs are used for RNA detection, then multiplexed detection capability is enhanced, but reagent requirements increase

Engineering Contradiction:

Improvemultiplexed detection capabilityVSAvoidreagent requirements

Core Design Contradiction:

Adaptability or versatilityVSQuantity of substance

Solution Approach 1:

The patent segments the detection system into a universal nuclease component and multiple specific tracrRNA components. Each tracrRNA is designed to detect a specific RNA target, allowing multiplexed detection where multiple tracrRNAs can work simultaneously with a single nuclease enzyme, reducing the total reagent quantity needed compared to using separate nucleases for each target

Inventive Principle:

Principle #1Segmentation

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 allows for sequence-specific detection of RNAs, providing a robust and versatile diagnostic tool with reduced off-targeting, capable of multiplexed RNA detection and transcriptional recording, enhancing the sensitivity and specificity of RNA detection in various applications.

Implementation Method 1

at least one non-naturally occurring tracrRNA specifically hybridizing with said sensed RNA

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

at least one tracrRNA-dependent CRISPR nuclease enzyme binding to at least one target nucleic acid

Methodology Applied

Scientific EffectSequence-specific binding:

Implementation Method 3

capable of cleaving a double-stranded nucleic acid molecule

Methodology Applied

Scientific EffectNuclease cleavage: Enzyme

Data Source

PatentUS20230175078A1RNA detection and transcription-dependent editing with reprogrammed tracrrnas

Publication Date: 2023.06.08 HELMHOLTZ ZENTRUM FUER INFEKTIONSFORSCHUNG GMBH

AI summary

The present invention relates to methods for detecting at least one sensed RNA in a cell, tissue, and/or sample using at least one non-naturally occurring tracrRNA specifically hybridizing with said sensed RNA and at least one tracrRNA-dependent CRISPR nuclease enzyme binding to at least one target nucleic acid, as well as respective systems and diagnostic and therapeutic uses thereof.