CRISPR-Cas12a One-Pot miRNA Detection via RCA
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Solution Overview
Problem
Current methods for detecting microRNAs (miRNAs) are challenging due to their short length, high sequence similarity, and wide concentration range, leading to issues with sensitivity, specificity, and ease of use, especially in clinical samples.
Innovation Solution
A one-pot CRISPR-Cas12a system that combines rolling circle amplification (RCA) with CRISPR-mediated detection, using a padlock probe and a ribonucleoprotein complex (RNP) to achieve exponential amplification and sensitive detection of miRNAs in a single isothermal reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional RT-qPCR is used for miRNA detection, then detection sensitivity and accuracy are improved, but the process complexity and instrument requirements increase
Solution Approach 1:
The patent combines reverse transcription, rolling circle amplification, and CRISPR-Cas12a detection into a single integrated reaction system. The padlock probe design allows RT and RCA to occur in the same pot, eliminating the need for separate amplification and detection instruments, thereby reducing device complexity while maintaining detection sensitivity.
Solution Approach 2:
The CRISPR-Cas12a system serves multiple functions: it provides sequence-specific recognition of the amplified miRNA target, enables exponential amplification through collateral cleavage activity, and generates detectable fluorescent signals. This multi-functionality replaces the need for separate amplification and detection systems required by traditional RT-qPCR.
2Measurement precision
If two-step RT-qPCR process is used, then detection accuracy is improved, but sample loss and contamination risk increase
Solution Approach 1:
The patent merges the reverse transcription step and amplification step into a single isothermal reaction vessel. The padlock probe remains hybridized to the miRNA target during RT, and the circularized probe serves as the template for RCA, eliminating the need for liquid transfer between steps and thus preventing contamination while maintaining detection accuracy.
3Ease of operation
If rolling circle amplification is used, then isothermal operation is achieved, but amplification efficiency and sensitivity are reduced
Solution Approach 1:
The patent introduces CRISPR-Cas12a as an intermediary that recognizes the RCA-amplified padlock probe sequence and catalyzes exponential amplification through its endonuclease activity. The Cas12a enzyme cleaves the RCA product to generate new padlock probes, creating a feedback loop that transforms linear RCA into exponential amplification while maintaining isothermal conditions.
4Measurement precision
If padlock probe with nick is used, then miRNA target recognition is improved, but ligation efficiency may be reduced
Solution Approach 1:
The padlock probe is designed with a nick at a specific location that is complementary to the miRNA target. The nick position is strategically chosen to ensure optimal hybridization with the miRNA while providing sufficient distance from the ligation site to allow efficient joining by DNA ligase. This local optimization of the nick position resolves the contradiction between target recognition and ligation efficiency.
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 system achieves high sensitivity with a detection limit of single-digit femtomolar concentrations, single-nucleotide specificity, and rapid analysis, comparable to traditional RT-qPCR methods, while simplifying the workflow and eliminating the need for specialized instruments.
Implementation Method 1
a CRISPR-Cas12a complex wherein the crRNA is capable of hybridizing with the detection zone of the padlock probe
Implementation Method 2
the ligation zone comprising a polynucleotide sequence complementary to a microRNA target of interest
Implementation Method 3
a polymerase; and a reporter deoxyribonucleic acid (DNA) capable of producing a CRISPR-generated detectable signal
Data Source
AI summary
Described herein are methods, compositions, and kits relating to the mixed detection of one or more polynucleotides, in particular miRNAs. In certain aspects, methods, systems, compositions, and kits utilize Cas12a for polynucleotide detection, in particular miRNAs. In aspects the methods, systems, compositions, and kits utilize Cas12a for detection of polynucleotide targets in a single pot reaction.