CRISPR-Cas9 Ribonucleoprotein Complex for DNA Labeling
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Solution Overview
Problem
Current DNA detection methods, such as in situ hybridization, require DNA denaturation and extensive sample preparation, leading to specimen degradation and lengthy processing times, limiting their effectiveness for routine applications.
Innovation Solution
A method using a ribonucleoprotein complex comprising an unlabeled Cas protein, an unlabeled crRNA, and a labeled tracrRNA is applied to paraformaldehyde-fixed tissue sections, allowing for specific DNA sequence detection without denaturation and reducing sample preparation requirements, enabling faster and more robust detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA denaturation is performed to achieve complementary base pairing in in situ hybridization, then hybridization can occur, but the structure of the specimen is degraded
Solution Approach 1:
The patent changes the fundamental parameter of DNA accessibility by using CRISPR-Cas9 to create localized unwound regions at target sites, allowing probe access without global denaturation. This maintains specimen structure while enabling hybridization at specific locations through programmable Cas9-guided DNA unwinding.
Solution Approach 2:
The CRISPR-Cas9 complex acts as an intermediary that facilitates probe access to target DNA sequences. The Cas9 protein, guided by crRNA, creates localized access points in the chromatin structure, enabling subsequent probe hybridization without requiring harsh denaturation conditions that would damage the specimen.
2Measurement precision
If classical FISH procedures including hybridization and post-hybridization washes are performed, then specific DNA sequences can be detected, but the process requires hours to days
Solution Approach 1:
The CRISPR-Cas9 complex is pre-assembled and applied to the specimen before hybridization. The Cas9 protein locally unwinds the DNA at target sites in advance, creating immediate access for the probe without requiring lengthy hybridization incubation periods or post-hybridization washes, thereby dramatically reducing processing time.
Solution Approach 2:
The patent extracts and eliminates the time-consuming steps of classical FISH (extended hybridization incubation and post-hybridization washes) by using CRISPR-Cas9 to pre-open chromatin structures. This allows direct probe binding to exposed target sequences, removing the need for prolonged processing steps.
3Measurement precision
If extensive sample preparation is performed for CRISPR-Cas9-mediated in situ labeling, then labeling accuracy improves, but the simplicity and routine applicability of the method decreases
Solution Approach 1:
The patent merges the CRISPR-Cas9 genome editing machinery with in situ hybridization detection into a single integrated workflow. The Cas9-complexed probe is applied directly to fixed tissue sections, combining targeting precision with detection capability in one step, eliminating the need for separate, complex sample preparation procedures.
Solution Approach 2:
The CRISPR-Cas9 system provides universal functionality for DNA targeting and detection across different tissue types and sequences. The same basic protocol can be applied to any DNA target by simply changing the crRNA sequence, making the method broadly applicable without requiring extensive optimization or specialized sample preparation for each application.
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 method allows for simple, rapid, and robust detection of chromosomal and nuclear DNA sequences in tissue sections, preserving specimen structure and enabling simultaneous protein detection, thus avoiding the need for hazardous formamide and reducing wash times.
Implementation Method 1
an unlabeled crRNA hybridizing with the target DNA sequence
Implementation Method 2
a labeled tracrRNA hybridizing with the crRNA
Implementation Method 3
Cas9-RNA initiates DNA unwinding at the PAM-proximal region, followed by the directional formation of an R-loop
Implementation Method 4
the sample being a tissue section which has been fixed with a buffered 4% paraformaldehyde solution
Data Source
Figure 1a~1c
Figure 2a~2b
Figure 3a~3c
AI summary
The invention provides a fast and robust method for the detection of a target DNA sequence in a sample, wherein the sample is contacted with a ribonucleoprotein complex comprising an unlabeled Cas protein, an unlabeled crRNA hybridizing with the target DNA sequence and a labeled tracrRNA hybridizing with the crRNA under conditions where the target DNA in the sample is able to hybridize with the crRNA.