CRISPR Imaging With RNA Aptamer Recruitment for Single-Copy Loci
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Solution Overview
Problem
Current CRISPR-based imaging systems face challenges in achieving high signal-to-background ratio and resolution, particularly for non-repetitive genomic sequences, due to issues like high background signals and difficulties in simultaneous expression and binding of multiple sgRNAs.
Innovation Solution
A CRISPR-based imaging system utilizing engineered sgRNAs with RNA aptamers and multimerization peptides, combined with dCas9 and fluorescent proteins, to enhance signal amplification and specificity for non-repetitive genomic loci imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple fluorescent probes are used to target multiple adjacent sequences to obtain higher resolution, then imaging resolution is improved, but background signal increases and complexity increases
Solution Approach 1:
The patent divides the imaging system into separate functional modules: dCas9 for target binding, sgRNAs with RNA aptamers for signal recruitment, and fluorescent proteins for detection. This segmentation allows each component to perform its specific function efficiently, reducing interference between components and lowering background signal while maintaining high resolution.
Solution Approach 2:
The patent introduces RNA aptamers as intermediary molecules that bridge dCas9 and fluorescent proteins. The sgRNAs containing RNA aptamers recruit fluorescent proteins to the target site through specific binding, acting as a mediator that transfers the fluorescent signal to the precise location without requiring multiple fluorescent probes simultaneously, thus reducing background signal.
2Adaptability or versatility
If dCas9 is fused with fluorescent proteins to realize gene imaging in living cells, then live cell imaging capability is achieved, but background signal increases due to free fluorescently labeled dCas9
Solution Approach 1:
The patent extracts the fluorescent protein from direct fusion with dCas9, instead using a separate recruitment mechanism. The fluorescent proteins are not constitutively fused to dCas9 but are recruited only when needed through the sgRNA-RNA aptamer-fluorescent protein interaction, eliminating the background signal from free fluorescently labeled dCas9 while preserving live cell imaging capability.
Solution Approach 2:
The patent uses sgRNAs with RNA aptamers as intermediaries to recruit fluorescent proteins to the target site. This intermediary mechanism ensures that fluorescent proteins are only present at the target location when bound to the sgRNA-dCas9 complex, eliminating non-specific background signal while maintaining the ability to image genes in living cells.
3Measurement precision
If multiple sgRNAs are used to target different genomic loci, then imaging specificity is improved, but simultaneous expression and binding of multiple sgRNAs becomes difficult
Solution Approach 1:
The patent designs sgRNAs with universal RNA aptamer modules that can recruit the same fluorescent protein system regardless of the specific genomic target. This universal design allows multiple sgRNAs targeting different loci to be expressed and function simultaneously using the same recruitment mechanism, eliminating the difficulty of coordinating multiple different systems while maintaining high imaging specificity.
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 improved resolution and labeling of single-copy non-repetitive gene loci in living cells by reducing background noise and ensuring synchronized expression of multiple sgRNAs, thereby enhancing imaging clarity.
Implementation Method 1
a fluorescent protein, which are operably linked between each other in a manner that is not limited
Data Source
AI summary
Provided are a CRISPR-based imaging system and use thereof. The imaging system comprises: (1) a dCas9-expressing vector or a dCas9 protein; (2) an engineered sgRNA-expressing vector, the engineered sgRNA comprising: a sgRNA backbone containing n copies of RNA aptamer, and a sgRNA sequence specific for a target gene to be detected, wherein n is an integer greater than or equal to 2; and (3) a fusion protein-expressing vector, the fusion protein comprising: an RNA-binding motif specifically recognizing the RNA aptamer, a multimerization peptide and a fluorescent protein, which are operably linked to each other. The imaging system has improved resolution, and achieves labeling and imaging of non-repetitive sequence, especially labeling and imaging of non-repetitive sequence within single-copy gene loci in living cells.