Type V CRISPR Cas Proteins With tracrRNA For High Temperature Nucleic Acid Detection

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Solution Overview

Problem

Programmable nucleases, such as CRISPR/Cas proteins, face challenges in accuracy and effectiveness under challenging biological sample conditions like high viscosity and metal chelating, limiting their specificity and efficiency across various sample conditions.

Innovation Solution

The use of Type V CRISPR/Cas proteins coupled with engineered guide nucleic acids, specifically tracrRNA, which enhances stability and activity at high temperatures, enabling cis cleavage, transcollateral cleavage, and nickase activities for nucleic acid modification and detection.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If programmable nucleases are used under challenging biological sample conditions, then nucleic acid cleavage activity is achieved, but accuracy and effectiveness deteriorate due to high viscosity and metal chelating

Engineering Contradiction:

Improveaccuracy and effectivenessVSAvoidhigh viscosity and metal chelating effects

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent employs Type V CRISPR/Cas proteins which have been engineered to function optimally at elevated temperatures (45-65°C). By changing the operational temperature parameter, the system overcomes the detrimental effects of high viscosity and metal chelating in biological samples, maintaining high accuracy and effectiveness for nucleic acid detection and modification.

Inventive Principle:

Principle #35Parameter changes

2Stability of the object's composition

If Type V CRISPR/Cas proteins are used at high temperatures, then stability and cleavage activity are improved, but the complexity of maintaining optimal conditions increases

Engineering Contradiction:

Improvestability at high temperaturesVSAvoidtemperature control requirements

Core Design Contradiction:

Stability of the object's compositionVSDevice complexity

Solution Approach 1:

The Type V CRISPR/Cas protein system is designed to perform multiple functions (cis cleavage, transcollateral cleavage, and nickase activity) within a single protein complex that operates across a broad temperature range (45-65°C). This multi-functionality reduces the need for separate systems for different temperatures, simplifying overall system complexity while maintaining stability.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Measurement precision

If engineered guide nucleic acids with tracrRNA are used, then specificity and efficiency are improved, but the complexity of guide nucleic acid design increases

Engineering Contradiction:

ImprovespecificityVSAvoidguide nucleic acid structure

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent utilizes tracrRNA as an intermediary component that mediates between the crRNA (which provides target specificity) and the Cas protein (which provides cleavage activity). This intermediary structure simplifies the overall design by separating the specificity-determining region (crRNA) from the protein-interaction region (tracrRNA), allowing independent optimization of each component while maintaining high specificity and efficiency.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 solution provides stable and efficient nucleic acid modification and detection capabilities across a wide range of conditions, including high temperatures, improving the accuracy and effectiveness of programmable nucleases in diagnostic and therapeutic applications.

Implementation Method 1

guide nucleic acids comprise a CRISPR RNA (crRNA) that is at least partially complementary to a target nucleic acid

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide RNA (crRNA or sgRNA), wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to guideRNA

Methodology Applied

Scientific EffectCis cleavage:

Implementation Method 3

Trans cleavage activity (also referred to as transcollateral cleavage) is cleavage of ssDNA or ssRNA that is near, but not hybridized to the guide RNA

Methodology Applied

Scientific EffectTrans collaterals cleavage:

Implementation Method 4

Nickase activity is the selective cleavage of one strand of a dsDNA molecule

Methodology Applied

Scientific EffectNickase activity:

Implementation Method 5

compositions and systems comprise a tracrRNA that renders the compositions stable and capable of cleaving nucleic acids at high temperatures

Methodology Applied

Scientific EffectThermal stability enhancement:

Data Source

PatentUS20240191280A1Enhanced guide nucleic acids and methods of use

Publication Date: 2024.06.13 MAMMOTH BIOSCIENCES INC

AI summary

The present disclosure describes CRISPR/Cas proteins and guide RNA combinations that are capable of modifying nucleic acids at high temperatures. These compositions are especially useful for systems and methods of nucleic acid detection, including diagnostic devices.