Self-reporting transposon constructs for single-cell mapping
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Solution Overview
Problem
Current methods lack the ability to simultaneously map transposon insertions and transcription factor binding sites at single-cell resolution, limiting the understanding of cellular heterogeneity and transcriptional regulation in complex tissues.
Innovation Solution
Development of self-reporting transposons (SRTs) that integrate into the genome and report their location via mRNA transcription, allowing for the simultaneous measurement of transposon insertions and mRNA abundance from single cells using advanced sequencing technologies, and a novel single-cell calling card method to identify cell types and transcription factor binding sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If bulk tissue analysis methods are used, then throughput is improved, but single-cell resolution is lost
Solution Approach 1:
The invention segments the bulk tissue into individual single cells for analysis. By isolating and analyzing thousands of individual cells separately, the method achieves both single-cell resolution (measurement precision) and high throughput (productivity) simultaneously, resolving the contradiction between bulk analysis throughput and single-cell precision.
2Measurement precision
If transposon insertion mapping is performed, then insertion locations are identified, but transcription factor binding sites cannot be simultaneously mapped
Solution Approach 1:
The invention merges transposon insertion mapping with transcription factor binding site mapping into a single integrated assay. By combining these two previously separate mapping techniques, the method enables simultaneous identification of both insertion locations and transcription factor binding sites at single-cell resolution, resolving the contradiction between specialized mapping precision and multi-functionality.
Solution Approach 2:
The method creates a universal platform that performs multiple mapping functions simultaneously. The single-cell sequencing approach can identify transposon insertions, transcription factor binding sites, and cellular heterogeneity all in one experiment, making the system adaptable to various mapping needs without requiring separate specialized assays.
3Measurement precision
If advanced sequencing technologies are used, then mapping precision is improved, but complexity of the method increases
Solution Approach 1:
The method employs self-reporting transposons that automatically provide their own location information through mRNA transcription. The transposons carry their own genomic coordinates in the form of flanking sequence transcripts, eliminating the need for complex external mapping procedures and reducing methodological complexity while maintaining high mapping precision.
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
Enables precise mapping of transposon insertions and transcription factor binding sites across thousands of single cells, providing insights into cellular diversity and regulatory elements, and allowing for lineage tracing and improved transposon mutagenesis screens.
Implementation Method 1
the promoter is capable of driving transcription of RNA through at least one transposon end after the SRT construct is inserted into genomic DNA, so that a portion of the transposon DNA, at least one transposon end, and the genomic DNA flanking the transposon end is transcribed into RNA
Implementation Method 2
the transcript can be recovered by reverse transcription using a poly-T primer
Data Source
AI summary
Among the various aspects of the present disclosure is the provision of compositions and methods for mapping transposon insertions. Applications can include mapping the locations of self-reporting transposons (SRTs) from thousands of single cells in parallel, while simultaneously measuring mRNA abundance from the same single cells; analyzing genome-associated protein (GAP) (e.g., transcription factor) binding/interactions in a small number of cells in bulk, without single cell resolution; lineage tracing; or as an improved readout for transposon mutagenesis screens.