CRISPR Barcode Cell Tagging for Viable Evolution Tracking
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Solution Overview
Problem
Current methods for tracking cell evolution are costly, destructive to cell viability, and lack the ability to scale for phenotypic and genomic characterization, making it difficult to study the determinants of evolutionary selection and clonal non-selection in cell populations.
Innovation Solution
A method using CRISPR-Cas guide molecules with barcodes to tag and isolate specific cell populations, enabling the tracking and recovery of viable cells through frameshift reporters, allowing for phenotypic and genomic characterization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-cell phenotypic and genetic characterization methods are used, then cell evolution can be tracked, but the cost increases and cell viability is lost
Solution Approach 1:
The patent uses DNA barcodes as copies of cell identity information. Instead of directly analyzing and destroying the original cells through sequencing, the method creates molecular copies (barcodes) that can be amplified and sequenced without touching the original viable cells. This allows repeated measurements of cell lineage and phenotype while cells remain alive and functional.
Solution Approach 2:
The patent introduces DNA barcodes as intermediary molecules between the cell and the analysis system. These barcodes serve as mediators that carry cell identity information and can be detected through sequencing without directly observing or damaging the cell itself. The barcode acts as a proxy that enables indirect measurement of cell properties.
2Measurement precision
If single-cell characterization methods are used, then phenotypic and genetic analysis is enabled, but the labor and cost increase significantly
Solution Approach 1:
The patent creates simplified molecular copies (DNA barcodes) that encapsulate cell identity information. Instead of performing complex single-cell sequencing and phenotypic analysis for each cell, the method uses these compact barcode copies that can be rapidly amplified and sequenced using standard high-throughput sequencing techniques, dramatically reducing computational and laboratory complexity.
Solution Approach 2:
The patent segments the cell analysis problem into two parts: (1) assigning a simple DNA barcode to each cell during proliferation, and (2) sequencing these barcodes to identify cell lineages. This segmentation separates the complex cell biology from the simple molecular sequencing, making the overall system more manageable and cost-effective.
3Measurement precision
If current barcoding strategies are used, then cell tracking is enabled, but phenotypic and genetic characterization is precluded
Solution Approach 1:
The patent makes the DNA barcode system universal by designing barcodes that can be simultaneously used for multiple purposes: (1) tracking cell lineages through proliferation, (2) enabling phenotypic characterization through associated reporter genes, and (3) facilitating genetic analysis through CRISPR-Cas9 integration. This multi-functional barcode system eliminates the need for separate tracking and characterization methods.
Solution Approach 2:
The patent merges previously separate functions into a single integrated system: cell tracking barcodes are combined with phenotypic reporters and genetic modification targets. This consolidation allows one molecular tool to simultaneously provide lineage information, phenotypic data, and genetic manipulation capability, greatly enhancing versatility.
4Measurement precision
If cells are isolated for study, then phenotypic and genomic analysis is possible, but the original population cannot be preserved
Solution Approach 1:
The patent uses DNA barcodes as molecular copies that can be sequenced without removing or damaging the original cells. The barcode information is copied from the cell's DNA and amplified through PCR and sequencing processes, allowing repeated analysis of the same cell population without depleting it. This enables both detailed cell analysis and population preservation simultaneously.
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 simultaneous tracking and isolation of specific cell populations, facilitating the elucidation of evolutionary selection processes and the molecular basis of phenotypic changes, including tumor formation and drug sensitivity.
Implementation Method 1
CRISPR-Cas guide molecules with barcodes to tag and isolate specific cell populations
Implementation Method 2
frameshift reporters, allowing for phenotypic and genomic characterization
Data Source
AI summary
The subject matter disclosed herein is generally directed to methods and compositions for tagging cells of interest, tracking evolution of the tagged cells, and recovering the original tagged cells for further study. Specifically, cells are tagged with a DNA construct encoding a barcode sequence comprising a guide sequence. Barcoded cells can then be recovered using a reporter construct having CRISPR target sequences specific for the cell having a barcode of interest.