Engineered Transposase for Antibody-Free Chromatin Sequencing
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Solution Overview
Problem
Current methodologies for sequencing chromatin fragments, such as ChIP-seq and Nextera, face limitations including low signals, high backgrounds, epitope masking, and reliance on antibodies, which restrict their suitability for multiplex sequencing and comprehensive analysis of epigenetic modifications, especially at the single-cell level.
Innovation Solution
Development of engineered transposases that can bind specifically to chromatin components like methylated histones, enabling the GET-seq and scGET-seq methods for comprehensive genomic and epigenomic analysis without the need for antibodies, allowing for simultaneous profiling of genomic, epigenomic, and transcriptomic data at the single-cell level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional ChIP-seq methodology is used, then chromatin fragments can be sequenced, but the process is complex, time-consuming, and requires large numbers of cells with low signals and high backgrounds
Solution Approach 1:
The invention extracts and eliminates the antibody-dependent steps from the chromatin sequencing workflow. By using engineered transposases that directly target chromatin modifications without requiring antibodies, the method removes complex immunoprecipitation steps, crosslinking procedures, and antibody optimization requirements, thereby simplifying the overall process while improving productivity
Solution Approach 2:
The engineered transposase system provides universal functionality for targeting multiple chromatin modifications simultaneously. A single transposase platform can be directed to different histone modifications through engineered specificity, eliminating the need for separate antibody-based protocols for each modification type, thus reducing complexity and enabling high-throughput multiplexed sequencing
2Measurement precision
If antibody-based approaches are used, then specific chromatin regions can be targeted, but epitope masking and technical challenges associated with antibodies occur
Solution Approach 1:
The invention introduces engineered transposases as intermediary molecules that directly recognize and bind to chromatin modifications. These transposases serve as reliable mediators between the sequencing apparatus and chromatin targets, eliminating the need for antibodies as intermediaries. The transposases provide consistent, antibody-independent targeting without epitope masking issues
Solution Approach 2:
The invention substitutes the biological recognition mechanism of antibodies with an engineered enzymatic recognition system. The transposase-based approach replaces antibody-antigen binding with transposase-chromatin modification interactions, providing more reliable and consistent targeting that is not subject to antibody limitations such as epitope masking, batch variability, and storage requirements
3Ease of manufacture
If traditional ligation-based library construction is used, then DNA libraries can be prepared, but the process is time-consuming and less efficient
Solution Approach 1:
The engineered transposase system performs library construction in a self-service manner. The transposase simultaneously fragments the chromatin, adds sequencing adapters, and prepares the library for sequencing in a single enzymatic reaction step. This self-contained approach eliminates the need for separate ligation, purification, and preparation steps required by traditional methods, dramatically reducing turnaround time while simplifying the manufacturing process
4Loss of information
If single-cell sequencing approaches are used, then cancer heterogeneity can be captured, but the current methods have limited multiplexing capabilities
Solution Approach 1:
The engineered transposase platform provides universal adaptability for multiplexed single-cell sequencing. By engineering transposases with different specificities for various chromatin modifications, multiple epigenetic markers can be simultaneously targeted in single-cell populations. This enables comprehensive capture of cancer heterogeneity across multiple epigenetic dimensions without requiring separate experiments for each marker
Solution Approach 2:
The invention segments the sequencing approach by enabling independent targeting of different chromatin modification types through specifically engineered transposases. This segmentation allows parallel analysis of multiple epigenetic features across single-cell populations, providing detailed resolution of cancer heterogeneity while maintaining the ability to perform multiplexed sequencing in a unified platform
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
These methods provide more efficient, robust, and cost-effective multiplex sequencing capabilities, enabling dynamic and comprehensive profiling of chromatin states and epigenetic modifications, including those previously inaccessible, thereby improving our understanding of cellular physiology and disease mechanisms.
Implementation Method 1
engineered transposases that can bind specifically to chromatin components like methylated histones
Implementation Method 2
the target dsDNA undergoes tagmentation by the transposase. Thus, the target dsDNA is fragmented and the transposon (including the ME and the sequencing primer) is covalently attached to the 5′ end of the target dsDNA fragment
Data Source
AI summary
The present invention relates to an engineered transposase comprising a transposase operably linked to a polypeptide that binds to a component of heterochromatin. The present invention further relates to an engineered transposome complex comprising an oligonucleotide and an engineered transposase according to the invention. The present invention also relates to methods and uses of the engineered transposase of the invention and engineered transposome of the invention for making a DNA sequence library or libraries and for DNA sequencing.