Cas12 Chimeric Probe Cleavage for Sensitive RNA Detection
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Solution Overview
Problem
Current CRISPR/Cas12 systems face challenges in effectively utilizing the RNase activity of Cas12 proteins for nucleic acid detection, particularly in trans-cleaving RNA sequences, limiting their application in diagnostic fields.
Innovation Solution
The method involves using Cas12 proteins to trans-cleave non-natural sequences, specifically sequences with both deoxynucleotides and ribonucleotides, or sequences with artificially created modifications, to enhance detection efficiency, including the use of chimeric sequences like rUArUArUA and ArUArUArU, which are cleaved by Cas12 proteins like LbCas12a and AsCas12a with improved efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If Cas12 proteins are used to trans-cleave natural RNA sequences, then RNA detection capability is improved, but cleavage efficiency is insufficient
Solution Approach 1:
The patent modifies the natural RNA sequence parameters by introducing non-natural modifications (such as 2'-O-methyl, phosphorothioate linkages) to create optimized probe sequences that enhance Cas12 cleavage efficiency while maintaining RNA detection capability
Solution Approach 2:
The patent creates composite nucleic acid probes combining natural and non-natural nucleotides, merging the advantages of RNA detection capability with improved stability and cleavage efficiency through non-natural sequence modifications
2Productivity
If non-natural sequences with artificial modifications are used as probes, then cleavage efficiency is improved, but probe design complexity increases
Solution Approach 1:
The patent systematically varies parameters of non-natural modifications (position, type, frequency) to identify optimal probe sequences that achieve high cleavage efficiency without requiring overly complex design procedures
Solution Approach 2:
The patent applies non-natural modifications at specific local positions within the probe sequence rather than uniformly throughout, optimizing cleavage efficiency at critical regions while simplifying overall probe design
3Device complexity
If conventional DNA probes are used for detection, then detection system simplicity is maintained, but signal-to-noise ratio is limited
Solution Approach 1:
The patent develops chimeric probes combining DNA and non-natural nucleotide components, achieving enhanced signal-to-noise ratio while maintaining compatibility with conventional Cas12 detection systems
Solution Approach 2:
The patent creates modified probe versions that replicate the functionality of conventional DNA probes while incorporating non-natural sequences to enhance detection precision and signal characteristics
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 accurate and sensitive detection of nucleic acids, including RNA, with enhanced signal-to-noise ratio and stability, expanding the applicability of Cas12 proteins in nucleic acid detection systems, and enabling high-throughput and clinical testing.
Implementation Method 1
the fluorescent group at one end and a fluorescent quenching group at the other end to reflect whether the Cas protein detects the presence of the target nucleic acid under the guidance of the crRNA. Furthermore, due to the high programmability of the crRNA, it can be used for guiding the Cas protein to target any sequence of interest. Under normal circumstances, the fluorescence emitted by the fluorescent group is quenched by the quenching group since the fluorescent group is close to the quenching group. However, when the Cas protein identifies the target nucleic acid under the guidance of the crRNA, it trans-cleaves the probe, so that the fluorescent group on the probe is separated from the quenching group, and the fluorescent group normally emits fluorescence, which is detected by an instrument, thereby achieving the purpose of indirect detection of the target nucleic acid.
Data Source
AI summary
A method for detecting a target nucleic acid includes cleaving a non-natural sequence using a Cas12 protein. The Cas12 protein belongs to a Cas12a/Cas12b protein family. The non-natural sequence includes a chimeric sequence with a ribonucleotide and a deoxyribonucleotide, which can be made into a probe for nucleic acid detection, and its detection effect is better than a conventional ssRNA probe, and may be equivalent to or even better than an ssDNA probe. In addition, a system for detecting a target nucleic acid includes a chimeric sequence, a Cas12 protein and crRNA. Pre-amplification can increase the detection limit of the system to a single-molecule level. The Cas12-chimeric sequence detection system can be used not only on a microfluidic chip, but also in clinical testing. The coordinated use of the Cas12 protein and chimeric sequence optimizes the CRISPR/Cas12 detection system and expands use of Cas12a protein and non-natural sequence.