Targeted In Situ Genome-Wide Profiling by Nuclease Cleavage
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Solution Overview
Problem
Current chromatin immunoprecipitation (ChIP) methods, such as ChIP-seq and ChIP-chip, suffer from biases and inefficiencies, particularly when dealing with limited cell numbers or small tissue samples, leading to incomplete extraction of protein-DNA complexes and high non-specific background noise, making them unsuitable for many applications.
Innovation Solution
A method involving a permeabilized cell or nucleus immobilized on a solid surface, treated with a specific binding agent coupled to an inactive nuclease, followed by activation to cleave DNA bound to the target protein, allowing excision and sequencing of the bound DNA, which can be performed on as few as 1 to 10,000 cells using single cell nanowell indexing or split pool combinatorial indexing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If chromatin immunoprecipitation (ChIP) is performed with formaldehyde crosslinking to preserve in vivo pattern, then mapping resolution is improved, but epitope masking and false positive binding sites increase
Solution Approach 1:
The patent extracts only the DNA bound to the protein of interest through nuclease cleavage at the protein-DNA interface, rather than extracting all chromatin through formaldehyde crosslinking. This selective extraction approach removes the harmful crosslinking step while preserving mapping resolution by directly isolating protein-bound DNA sequences.
Solution Approach 2:
The patent introduces a nuclease as an intermediary enzyme that cleaves DNA at specific locations where it is bound to the protein of interest. This intermediary mechanism replaces formaldehyde crosslinking and enables precise DNA release without causing epitope masking or false positive binding sites.
2Adaptability or versatility
If chromatin is fragmented and solubilized for immunoprecipitation to enable genome-wide mapping, then mapping coverage is improved, but non-specific background increases
Solution Approach 1:
The patent applies local quality by performing nuclease cleavage only at specific locations where the protein of interest binds to DNA, rather than fragmenting and solubilizing all chromatin. This localized approach maintains genome-wide mapping coverage through in situ processing while eliminating non-specific background by preventing random DNA release.
Solution Approach 2:
The patent performs preliminary action by immobilizing cells or nuclei on a solid surface and introducing the protein of interest onto the chromatin before nuclease addition. This preliminary setup ensures that only DNA bound to the specific protein is subsequently cleaved and released, maintaining mapping coverage while preventing non-specific background.
3Object-affected harmful factors
If ChIP is performed without crosslinking to minimize epitope masking, then extraction efficiency is improved, but protein-DNA complex extraction completeness decreases
Solution Approach 1:
The patent replaces the mechanical/chemical system of formaldehyde crosslinking with an enzymatic system using nucleases. This substitution eliminates epitope masking while improving extraction efficiency because the nuclease directly cleaves DNA at protein-binding sites, releasing complete protein-DNA complexes without requiring crosslinking.
4Reliability
If ChIP requires large numbers of cells to achieve sufficient signal, then mapping reliability is improved, but applicability to limited cell numbers decreases
Solution Approach 1:
The patent applies self-service by performing all chromatin processing steps in situ within the cell or nucleus, eliminating the need for extensive chromatin fragmentation and solubilization that requires large cell numbers. The nuclease cleaves DNA directly at protein-binding sites within intact cells, enabling reliable mapping with limited cell numbers while maintaining mapping reliability.
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 provides high-resolution mapping of protein-DNA interactions with reduced background noise and improved efficiency, enabling accurate profiling of chromatin-associated factors even with limited sample quantities.
Implementation Method 1
activating the nuclease and cleaving DNA bound to the chromatin-associated factor of interest to thereby excise the DNA bound to the chromatin-associated factor of interest
Implementation Method 2
the cell and/or nucleus is permeabilized by contacting the cell with digitonin
Data Source
Figure 1A~1D
Figure 2A~2B
Figure 3A~3C
AI summary
A method for detecting the binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in a cell, including: contacting a permeabilized cell or nucleus with a specific binding agent that specifically recognizes the chromatin-associated factor of interest, wherein the specific binding agent is linked to a nuclease that is inactive or an activatable transposome; activating the nuclease or transposase, thereby excising the sequence of chromatin DNA bound to the chromatin-associated factor of interest; isolating the excised DNA; and determining the sequence of the excised DNA, thereby detecting binding of a chromatin-associated factor of interest to a sequence of chromatin DNA in the cell.