Single-Cell Multi-Omics Analysis via Index Sorting and Barcoding
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Solution Overview
Problem
Current techniques lack the capability to effectively analyze both the genome and transcriptome simultaneously at the single-cell level, particularly for characterizing CRISPR editing outcomes and phenotypes in primary human cells.
Innovation Solution
A method involving labeling cells with detectable antibodies, index sorting, and subsequent processing with oligo-conjugated antibodies, reverse transcriptase, and specific primers to amplify and sequence genomic DNA, cDNA, and antibody-derived tags (ADTs) from individual cells, allowing for concurrent characterization of genomic and transcriptomic changes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current techniques are used to analyze genome and transcriptome, then analysis can be performed separately, but simultaneous single-cell analysis of both genome and transcriptome is not achievable
Solution Approach 1:
The patent combines genome amplification and transcriptome amplification into a single unified workflow. Both DNA and RNA are amplified simultaneously using shared reagents (reverse transcriptase, polymerase, dNTPs) and sequential PCR steps, enabling concurrent analysis of both genomes in the same single-cell reaction system rather than requiring separate analysis pipelines
Solution Approach 2:
The patent employs universal reagents that perform multiple functions: reverse transcriptase converts both RNA to cDNA and can also act as a polymerase for DNA amplification; the same PCR handles, barcodes, and sequencing primers are used for both genomic and transcriptomic libraries. This multi-functionality reduces system complexity while enabling simultaneous analysis of both data types
2Measurement precision
If single-cell analysis methods are developed to characterize CRISPR editing outcomes, then genomic and transcriptomic variation can be revealed, but the techniques remain technically challenging and lack scalability
Solution Approach 1:
The patent incorporates index sorting and cell barcoding before amplification, allowing cells to be pre-labeled with unique identifiers and sorted into individual wells or droplets. This preliminary action enables subsequent high-throughput processing of thousands of cells in parallel, maintaining single-cell resolution while achieving scalability through pre-organized sample architecture
Solution Approach 2:
The patent uses molecular barcodes and UMIs (unique molecular identifiers) as digital copies that track individual molecules and cells through the amplification and sequencing process. These barcode copies allow computational deconvolution of pooled samples, enabling precise tracking of CRISPR editing outcomes in each original cell even when processed in large batches, thus achieving both precision and scalability
3Loss of information
If existing methods are used for polynucleotide sequence alteration analysis, then some characterization is possible, but correlative relationships between genomic and transcriptomic changes cannot be established
Solution Approach 1:
The patent segments the analysis into distinct but coordinated library preparation pathways: one for genomic DNA amplification and one for cDNA amplification from the same cell lysate. Each pathway uses cell-specific barcodes to tag molecules, allowing the segmented analyses to be computationally re-integrated to reveal correlations between genomic alterations and transcriptomic changes while maintaining manageable complexity in each individual workflow
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 robust and scalable analysis of tens of thousands of cells, correlating genomic editing with mRNA and protein expression, and characterizing alterations in CRISPR-edited cells, overcoming limitations of existing methods that struggle with scalability and reliability in single-cell analysis.
Implementation Method 1
labeling cells with detectable antibodies that specifically binds a cell surface marker of interest
Implementation Method 2
incubating the product of (c) with reverse transcriptase, a custom template switch oligo (TSO) containing one member of a binding pair, under conditions that permit generation of cDNA
Implementation Method 3
incubating the product of step (d) with genomic primers that specifically bind a region of interest (ROI), cDNA amplification primers that specifically bind the PCR handle and the TSO, an antibody derived tag (ADT) specific primer, dNTPs, and a polymerase under conditions that support amplification
Data Source
AI summary
The disclosure provides compositions and methods for characterizing the genome and transcriptome at a single cell level. In some embodiments, the method provides for the characterization of CRISPR editing outcomes and phenotypes, as well as other alterations in polynucleotide sequences, particularly in primary cells.