Split Cas Protein Detection for Non-Nucleic Acid Targets
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Solution Overview
Problem
Existing CRISPR/Cas12a biosensing systems face challenges in transforming into a universal and simple detection system for non-nucleic acid analytes, with limitations such as heterogeneity, long experimental cycles, and complex operations, especially when replacing traditional ELISA kits.
Innovation Solution
A detection system utilizing split CRISPR/Cas proteins with specific fusion proteins, linker peptides, and guide RNA for flexible receptor adaptation, enabling homogeneous, highly sensitive, and rapid visualizable detection of non-nucleic acid targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional ELISA kits are replaced with CRISPR/Cas12a biosensing systems for non-nucleic acid detection, then detection sensitivity and specificity are improved, but system complexity and operational complexity increase
Solution Approach 1:
The Cas12a protein is divided into two separate fragments (N-terminal fragment and C-terminal fragment) that are expressed independently. These fragments are then brought into proximity through specific binding interactions with the target analyte, allowing the system to achieve high sensitivity while maintaining modularity and reducing overall system complexity through component separation.
Solution Approach 2:
A bridge molecule or complex is introduced as an intermediary that connects the two Cas12a fragments. This intermediary facilitates the reassembly of functional Cas12a activity only when the target analyte is present, enabling sensitive detection while simplifying the operational workflow by providing a clear on/off signal mechanism.
2Adaptability or versatility
If CRISPR/Cas12a system is adapted for non-nucleic acid detection, then application range is expanded, but experimental cycle time increases
Solution Approach 1:
The two Cas12a fragments are pre-expressed and pre-prepared in advance, and the binding components (antibodies, aptamers, or other target-specific binders) are pre-assembled. When the target analyte is introduced, the system rapidly assembles the complete detection complex without requiring time-consuming expression or assembly steps during the actual detection process.
Solution Approach 2:
The detection system is designed to automatically assemble the functional Cas12a complex through self-organizing interactions between the fragments and target-specific binders when the analyte is present. This self-assembly mechanism eliminates the need for complex manual manipulation or lengthy optimization procedures, reducing experimental cycle time while maintaining versatility across different target types.
3Ease of operation
If split Cas protein system is used for non-nucleic acid detection, then operational simplicity is improved, but detection precision may be compromised
Solution Approach 1:
The two Cas12a fragments are designed with specific local features (such as surface-exposed residues, charged patches, or structural motifs) that enable selective and high-affinity interactions with the target analyte and bridge molecules. These localized interaction interfaces ensure precise target recognition and binding, maintaining detection precision while allowing the rest of the system to remain simple and easy to operate.
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 nanogram-level detection with high specificity and rapid visual confirmation of non-nucleic acid targets, overcoming the limitations of traditional methods by providing a flexible and efficient detection method.
Implementation Method 1
a guide RNA, wherein the guide RNA guides the Cas protein to specifically bind to the Cas protein cis-cleavage substrate or the Cas protein cis-cleavage reporter
Implementation Method 2
a Cas protein cis-cleavage substrate or a Cas protein cis-cleavage reporter
Data Source
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AI summary
Provided are a non-nucleic acid target detection method based on a split Cas protein and the use thereof. Specifically, provided is a detection system for detecting a non-nucleic acid target, which detection system comprises (a) a first fusion protein or a first binding protein, and a second fusion protein or a second binding protein; (b) a Cas protein cis-cleavage substrate or a Cas protein cis-cleavage reporter; and (c) a guide RNA. In the system, a receptor can be flexibly changed according to the non-nucleic acid target, so as to achieve detection of various non-nucleic acid targets. For the first instance, a split Cas protein is used to detect a non-nucleic acid target, which has the characteristics of high levels of sensitivity, strong specificity and a short consumption time.