Trans-splicing Barcode Cassette for Transcriptome Profiling
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Solution Overview
Problem
Current methods for probing heterogeneity in cellular expression, such as sorting and isolating individual cells and extracting RNA, are limited and do not allow for rapid profiling of transcriptomes while retaining cell identities.
Innovation Solution
A method involving the delivery of a donor expression vector encoding a trans-splicing barcode cassette into cells, which includes an intron sequence followed by a splice-site and a barcode polynucleotide, allowing for the splicing of the barcode onto mRNA transcripts and subsequent identification of mRNA transcripts within a population of cells through high-throughput sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If individual cells are sorted and separated to profile transcriptomes, then cell identity information is retained, but the process becomes time-consuming and technically complex
Solution Approach 1:
The patent creates copies of cell identity information by integrating barcode sequences into the genome of individual cells. These barcodes serve as permanent identifiers that can be retrieved without manipulating the actual cells, enabling transcriptome profiling to be linked back to specific cell identities through sequencing rather than through complex sorting procedures
Solution Approach 2:
The patent performs preliminary action by integrating barcode sequences into cell genomes before transcriptome profiling. This pre-established identity tagging system allows for rapid association of transcriptome data with cell identities without requiring time-consuming cell sorting and isolation procedures at the time of profiling
2Measurement precision
If sufficient quantity and quality RNA is isolated from individual cells, then transcriptome profiling is accurate, but the process becomes complex and time-consuming
Solution Approach 1:
The patent creates copies of cell identity information through genomic barcode integration, allowing transcriptome data to be associated with cell identities through sequencing without requiring complex RNA isolation from individually sorted cells. The barcode copies serve as proxies for cell identity, simplifying the overall workflow
Solution Approach 2:
The patent merges cell identity information with transcriptome data by using the same barcode sequence for both purposes. Instead of separately maintaining cell identity records and transcriptome profiles, the system combines these functions into a unified approach where the barcode serves dual roles as both cell identifier and transcriptome tag
3Productivity
If rapid profiling of multiple cells is achieved, then productivity increases, but the ability to retain individual cell identities is lost
Solution Approach 1:
The patent creates copies of cell identity information in the form of genomic barcodes that can be rapidly retrieved through sequencing. This copying mechanism enables high-throughput profiling while preserving cell identity information, as the barcode sequences serve as permanent, easily accessible records of individual cell identities
Solution Approach 2:
The patent performs preliminary action by pre-integrating barcode sequences into cell genomes before high-throughput profiling. This advance preparation of identity tags enables rapid association of transcriptome data with cell identities during the profiling process, maintaining both speed and information retention
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 the rapid and efficient identification of mRNA transcripts in multiple cells, providing a means to profile transcriptomes and understand cellular heterogeneity, which is essential for studying biological systems and complex tissues like the brain or tumors.
Implementation Method 1
each of the at least two cells produces multiple copies of the trans-splicing barcode cassette encoded by the donor expression vector, which multiple copies of the trans-splicing barcode cassette each splice the barcode polynucleotide onto a mRNA transcript of the at least two cells
Data Source
AI summary
The present invention provides a method of identifying mRNA transcripts in the transcriptome of a cell comprising i) delivering into the cell a donor expression vector comprising nucleotides in a sequence encoding a trans-splicing barcode cassette, ii) exposing the cell to conditions such that the cell produces multiple copies of the trans-splicing barcode cassette encoded by the donor expression vector, which multiple copies of the trans-splicing barcode cassette each splice the barcode polynucleotide onto a mRNA transcript of the cell, thereby forming multiple mRNA transcripts of the cell, each spliced to the barcode polynucleotide; and iii) identifying the multiple mRNA transcripts that are spliced to the barcode polynucleotides, thereby identifying mRNA transcripts in the transcriptome of the cell.