Type V CRISPR-Cas ssDNA Cleavage for Sensitive DNA Detection
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Solution Overview
Problem
Existing CRISPR-Cas systems struggle to efficiently detect and cleave non-targeted single-stranded DNA (ssDNA) in samples, limiting their utility in sensitive and specific nucleic acid detection and manipulation.
Innovation Solution
Utilizing type V CRISPR/Cas proteins, such as Cas12a and Cas12b, which are activated by target DNA hybridization to promiscuously cleave non-targeted ssDNA, enabling detection through the use of a single-stranded detector DNA and measurement of cleavage signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing CRISPR-Cas systems are used to detect and cleave non-targeted ssDNA, then detection capability is limited, but system complexity and requirements for multiple components increase
Solution Approach 1:
The type V CRISPR-Cas effector protein performs multiple functions: it binds to guide RNA to form a surveillance complex, recognizes target DNA through base pairing, and promiscuously cleaves non-targeted ssDNA upon activation. This multi-functionality eliminates the need for separate detection and cleavage systems, reducing overall system complexity while enhancing detection sensitivity.
Solution Approach 2:
The effector protein automatically activates upon binding target DNA through guide RNA hybridization, and this activation inherently triggers the cleavage of detector ssDNA. The system serves itself by using the target recognition event to directly initiate the cleavage function without requiring external activation signals or additional control mechanisms.
2Measurement precision
If type V CRISPR-Cas proteins are used to promiscuously cleave non-targeted ssDNA, then detection sensitivity improves, but specificity to non-target sequences decreases
Solution Approach 1:
The guide RNA serves as an intermediary that provides sequence-specific recognition of the target DNA. The effector protein itself is non-specific for ssDNA cleavage, but the guide RNA-mediated target recognition ensures that cleavage only occurs when the correct target is present, thus maintaining specificity through the intermediary guide sequence.
Solution Approach 2:
The system exhibits different specificity characteristics at different functional stages: the guide RNA binding region provides high sequence specificity for target recognition, while the nuclease domain exhibits promiscuous (low specificity) cleavage activity toward ssDNA. This local differentiation of quality allows simultaneous achievement of specific target detection and sensitive signal generation.
3Reliability
If activation by target DNA hybridization is used, then detection accuracy improves, but response time increases due to hybridization requirements
Solution Approach 1:
The effector protein and guide RNA are pre-assembled into a surveillance complex before encountering the target DNA. This preliminary assembly ensures that upon target encounter, recognition and activation occur rapidly without requiring de novo complex formation, thus reducing response time while maintaining the accuracy of hybridization-based detection.
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
Enables highly sensitive and specific detection of target DNA, even at low concentrations, by leveraging the non-specific cleavage of ssDNA, facilitating rapid and accurate nucleic acid identification in various environments.
Implementation Method 1
base pairing between the guide RNA and a target sequence within the target nucleic acid molecule
Implementation Method 2
the protein becomes a nuclease that promiscuously cleaves ssDNAs
Data Source
AI summary
Provided are compositions and methods for detecting a target DNA (double stranded or single stranded) in a sample. In some embodiments, a subject method includes: (a) contacting the sample with: (i) a type V CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e); (ii) a guide RNA (comprising a region that binds to the type V CRISPR/Cas effector protein, and a guide sequence that hybridizes with the target DNA); and (iii) a detector DNA that is single stranded (i.e., a “single stranded detector DNA”) and does not hybridize with the guide sequence of the guide RNA; and (b) measuring a detectable signal produced by cleavage (by the type V CRISPR/Cas effector protein) of the single stranded detector DNA. Also provided are compositions and methods for cleaving single stranded DNAs (e.g., non-target ssDNAs), e.g., inside of a cell.