Cas12b Nucleic Acid Detection for Single-Nucleotide Specificity
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Solution Overview
Problem
Existing CRISPR-based nucleic acid detection platforms, such as those using Cas12a and Cas13, lack the necessary sensitivity and specificity for rapid and accurate detection of nucleic acids, particularly in clinical diagnostics and quarantine inspections.
Innovation Solution
A Cas12b-based nucleic acid detection method, termed CDetection, which enhances specificity and sensitivity to the attomolar level, allowing for the differentiation of nucleotide polymorphisms, and an enhanced version, eCDetection, which uses tuned guide RNAs (tgRNAs) for single-nucleotide resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Cas12a or Cas13 is used for nucleic acid detection, then the detection can be performed with existing platforms, but the sensitivity and specificity are insufficient for rapid and accurate detection
Solution Approach 1:
The patent changes the key parameter from the type of Cas enzyme used (Cas12a, Cas13) to a different Cas enzyme (Cas12b), which has distinct biochemical properties including higher specificity for DNA detection and the ability to distinguish single nucleotide polymorphisms. This parameter change resolves the contradiction by providing both high sensitivity and high reliability simultaneously
Solution Approach 2:
The patent segments the detection capability by using tuned guide RNAs (tgRNAs) with specific spacer sequences that can be designed to recognize exact target sequences including single nucleotide variations. This segmentation of recognition specificity allows the system to achieve both high sensitivity for target detection and high reliability by distinguishing even single nucleotide differences
2Measurement precision
If standard CRISPR detection platforms are used, then the detection process is established, but they cannot distinguish targets that differ by single nucleotide polymorphism
Solution Approach 1:
The patent applies local quality by designing tgRNAs where the spacer sequence (a local region of the guide RNA) is specifically tuned to match the target sequence including single nucleotide polymorphisms. This localized specificity in the spacer region enables nucleotide differentiation without requiring complex system-level changes, thus resolving the contradiction between measurement precision and device complexity
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
CDetection and eCDetection provide rapid and accurate DNA detection with high sensitivity and specificity, enabling applications in health and biotechnology, including ABO blood genotyping and mutation detection in human genes like BRCA1 and TP53.
Implementation Method 1
a gRNA directed against a target sequence in the target nucleic acid molecule
Implementation Method 2
a single-strand DNA reporter molecule which produces a detectable signal after being cleaved
Data Source
AI summary
A method for detecting the presence and/or amount of a target nucleic acid molecule in a biological sample and a related kit. The method comprises: (a) contacting the biological sample with: i) Cas12b protein, ii) a gRNA targeting a target sequence of the target nucleic acid molecule, and iii) a single-stranded DNA reporter molecule generating a detectable signal after being cleaved to form a reaction mixture; and (b) detecting the presence and/or level of the detectable signal generated in the reaction mixture.