Synthetic CRISPR Spacer Tags for Bacterial Labeling
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Solution Overview
Problem
Current CRISPR-Cas systems for bacterial immunity are not suitable for labeling and detecting specific bacterial cells without interfering with the cell's natural functions, as they are designed for sequence-specific targeting and interference, which can lead to self-targeting and erroneous behavior within the cellular machinery.
Innovation Solution
Development of synthetic CRISPR spacer polynucleotides and arrays that are non-functional, non-transcribable, and non-targeting, allowing them to be introduced into bacterial CRISPR arrays without triggering the immune response, enabling labeling and detection of specific bacteria by encoding a spacer tag that spells a text when translated, minimizing self-targeting risks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-Cas systems are used for bacterial immunity, then sequence-specific targeting capability is improved, but self-targeting and erroneous behavior occur
Solution Approach 1:
The invention extracts only the spacer component from the functional CRISPR-Cas system, creating a non-functional spacer polynucleotide that can be integrated into bacterial CRISPR arrays without the associated harmful targeting and interference activities. This allows the spacer to serve as a safe label without triggering self-targeting or erroneous cellular behavior.
Solution Approach 2:
The non-functional spacer polynucleotide acts as an intermediary element that provides the labeling function without mediating the harmful targeting and interference activities of the complete CRISPR-Cas system. It serves as a safe bridge between the desire for specific bacterial identification and the need to avoid cellular disruption.
2Reliability
If functional CRISPR spacers are integrated into bacterial CRISPR arrays, then adaptive immunity is improved, but cellular disruption occurs
Solution Approach 1:
The invention removes the functional components (cas genes, promoter regions, and other elements necessary for transcription and interference) from the CRISPR system, leaving only a non-functional spacer polynucleotide. This extracted spacer can be safely integrated into bacterial CRISPR arrays to provide labeling without disrupting cellular functions.
Solution Approach 2:
The non-functional spacer polynucleotide serves as a disposable labeling element that can be integrated into the CRISPR array without requiring the complex, energy-intensive, and potentially harmful functional CRISPR-Cas machinery. It provides the necessary identification function without the costly and disruptive biological activity.
3Adaptability or versatility
If synthetic CRISPR spacer polynucleotides are introduced for labeling, then bacterial identification capability is improved, but system complexity increases
Solution Approach 1:
The invention applies local quality by creating a specifically designed non-functional spacer polynucleotide with unique sequence characteristics (such as encoded text when translated) that provides identification capability at a localized level within the CRISPR array, without requiring complex system-wide changes to the bacterial cell.
Solution Approach 2:
The non-functional spacer polynucleotide serves as a simple copy or replica of the spacer component found in functional CRISPR systems, but without the complex associated machinery. This copying approach allows bacterial identification through sequence detection while avoiding the complexity of implementing the full functional CRISPR-Cas system.
Data Source
AI summary
This invention relates to synthetic CRISPR spacer polynucleotides and compositions comprising the same such as synthetic spacer-repeat sequences and synthetic CRISPR arrays, wherein the synthetic CRISPR spacer polynucleotides, when translated according to amino acid single-letter code convention, spell a text. Further provided in this invention are methods of using the same for labeling and/or detecting bacteria of interest.