CRISPR/Cas Proximity Detection for In Situ Nucleic Acid Imaging

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Solution Overview

Problem

Current fluorescent in situ hybridization (FISH) techniques for visualizing chromatin and RNA require harsh conditions that disrupt the integrity of chromatin structure and RNA-protein interactions, necessitating the development of milder methods for in situ imaging that provide robust signal amplification.

Innovation Solution

The use of RNA-guided nucleic acid binding proteins, specifically engineered CRISPR/Cas systems, to form proximity detection probe complexes that bind to endogenous nucleic acids, enabling visualization through proximity-dependent amplification reactions like PLA or proxHCR without denaturing chromosomal DNA.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If FISH uses harsh treatment to denature double-stranded genomic DNA for probe hybridization, then probe hybridization is achieved, but chromatin structure integrity and RNA-protein interactions are disrupted

Engineering Contradiction:

Improveprobe hybridizationVSAvoidchromatin structure integrity

Core Design Contradiction:

Measurement precisionVSStability of the object's composition

Solution Approach 1:

The patent changes the fundamental parameter of DNA denaturation from harsh chemical/thermal treatment to mild conditions by using CRISPR/Cas9 system that binds to double-stranded DNA in its native state. The guide RNA directs Cas9 to specific genomic loci without requiring DNA denaturation, thus maintaining chromatin structure integrity while achieving specific probe binding.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent replaces the mechanical/chemical denaturation process of traditional FISH with a biological recognition system. The CRISPR/Cas9 complex uses sequence-specific RNA-DNA hybridization to target genomic loci, substituting the need for harsh denaturation with a more gentle, sequence-specific binding mechanism that preserves chromatin architecture.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Measurement precision

If FISH uses harsh treatment to denature DNA, then probe binding is enabled, but RNA secondary structure and stability are affected

Engineering Contradiction:

Improveprobe bindingVSAvoidRNA secondary structure

Core Design Contradiction:

Measurement precisionVSStability of the object's composition

Solution Approach 1:

The patent changes the binding condition parameter from requiring denatured single-stranded DNA to binding on double-stranded DNA. The CRISPR/Cas9 system maintains DNA in its native double-stranded state during probing, which indirectly protects RNA secondary structures from disruption by harsh denaturation conditions.

Inventive Principle:

Principle #35Parameter changes

3Ease of operation

If FISH uses harsh treatment, then chromosomal regions become accessible for probing, but chromosomal organization is affected

Engineering Contradiction:

Improvechromosomal region accessibilityVSAvoidchromosomal organization

Core Design Contradiction:

Ease of operationVSStability of the object's composition

Solution Approach 1:

The patent replaces harsh mechanical/chemical treatment with a sequence-specific biological recognition system. The CRISPR/Cas9 complex can access and bind to specific chromosomal regions through guide RNA-directed targeting without disrupting the overall chromosomal organization, as it operates on double-stranded DNA in its native configuration.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

4Measurement precision

If signal amplification is increased in FISH, then detection sensitivity improves, but harsher treatment conditions are required

Engineering Contradiction:

Improvedetection sensitivityVSAvoidharsh treatment conditions

Core Design Contradiction:

Measurement precisionVSObject-affected harmful factors

Solution Approach 1:

The patent introduces signal amplification intermediaries that work under mild conditions. The CRISPR/Cas9 system itself provides amplification through the recruitment of multiple detection molecules to each target site, and additional amplification can be achieved through proximity-dependent enzymatic reactions or fluorescent tag multiplication without requiring harsh treatment conditions.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 sensitive and specific detection of endogenous nucleic acids in situ without disrupting chromatin or RNA structures, providing robust signal amplification and maintaining the integrity of cellular components.

Implementation Method 1

RNA-guided nucleic acid binding proteins, specifically engineered CRISPR/Cas systems, to form proximity detection probe complexes that bind to endogenous nucleic acids

Methodology Applied

Scientific EffectRNA-guided nucleic acid binding:

Implementation Method 2

enabling visualization through proximity-dependent amplification reactions like PLA or proxHCR

Methodology Applied

Scientific EffectProximity-dependent amplification:

Data Source

PatentUS11268144B2Methods and reagents for molecular proximity detection using RNA-guided nucleic acid binding proteins

Publication Date: 2022.03.08 EMD MILLIPORE CORP

AI summary

The present disclosure provides reagents and methods for molecular proximity detection of specific endogenous nucleic acids in situ using RNA-guided nucleic acid binding proteins.