Molecular Cell Diary System for Single-Cell Lineage Tracking
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Solution Overview
Problem
Current technologies are inadequate for real-time, dynamic recording of cellular events and lineage tracing at the single-cell level, particularly in understanding cell fate determination and tumor progression, due to limitations in resolution and the ability to discern individual cellular heterogeneity and molecular dynamics.
Innovation Solution
The Molecular Cell Diary System (MCDS) uses a combination of DNA cutters and writers to record specific genomic alterations linked to cellular events, allowing for the simultaneous assessment of lineage identity, cell divisions, and dynamic changes such as EMT initiation by writing short DNA sequences at double-strand breaks, which can be identified through next-generation sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional Omics and system biology methods are used to study cellular signaling, then vast amounts of data can be generated, but the data lacks direct biological relevance and creates more questions than answers
Solution Approach 1:
The invention segments the cellular signaling study into individual cell tracking by assigning unique barcodes to each cell. This allows the vast amount of data to be organized at the single-cell level, preserving direct biological relevance by tracking actual cellular lineages and fates rather than bulk population averages.
Solution Approach 2:
The invention creates a genetic copy (barcode) of each cell's identity that can be tracked through generations. This copying mechanism allows reconstruction of cellular histories and lineages, providing direct biological relevance to the data by maintaining a permanent record of each cell's origin and developmental path.
2Measurement precision
If terminally differentiated cells are studied to understand cell fate determination, then cell fate factors can be identified, but these factors are expressed transiently and early in differentiation making them poorly defined in many tissues
Solution Approach 1:
The invention performs preliminary action by tracking cells from their earliest stages through barcode assignment before fate determination occurs. This allows retrospective identification of cell fate factors and timing by analyzing the recorded cellular histories, capturing transient events that would be missed by studying only terminally differentiated cells.
Solution Approach 2:
The invention implements feedback by using the accumulated lineage data to inform and refine understanding of cell fate determination mechanisms. The tracked cellular histories provide continuous information about when and how fate factors are expressed, allowing researchers to identify transient expression patterns that would otherwise be lost.
3Productivity
If the average population approach is used to study cellular heterogeneity, then overall trends can be identified, but the detailed circuitry of individual fate choices is masked due to averaging
Solution Approach 1:
The invention segments the population data into individual cell lineages through unique barcode tracking. This allows simultaneous analysis of both overall trends (by aggregating barcode data across populations) and individual fate choices (by examining specific lineage histories), eliminating the masking effect of averaging while maintaining productivity through high-throughput barcode sequencing.
4Measurement precision
If rare early committed cells are isolated to determine lineage control, then potential fate factors can be identified, but the true fates become harder to ascertain after extraction from the system
Solution Approach 1:
The invention creates a permanent genetic copy (barcode) of each cell's identity before isolation. This allows the true fate of isolated rare cells to be ascertained by tracking their barcode through subsequent development and differentiation, maintaining reliability of fate determination even after extraction from the original system.
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
MCDS enables comprehensive, single-cell analysis of somatic alterations and cellular histories, providing detailed insights into cell lineage, timing, and molecular activity dynamics, which is crucial for understanding cancer progression and developing targeted therapies.
Implementation Method 1
a DNA cutter that creates double-strand breaks (DSBs) in the genome of the cell in a sequence specific manner
Implementation Method 2
The endogenous DNA synthetic and repair machineries, for example, Non-homologous End Joining (NHEJ) pathway, synthesize the complementary strand to the 'newly written' DNA sequence and seal the DSB
Data Source
AI summary
The subject invention pertains to a Molecular Cell Diary System (MCDS), which allows identification of the history of somatic alterations in the cell. MCDS comprises one or more combinations of a DNA cutter and a DNA writer expressed under the control of a promoter controlled a cellular event of interest. The DNA cutter and the DNA writer are in a combination are co-expressed when an even of interest occurs. The DNA cutter creates double strand breaks (DSB) in a target DNA in a sequence specific manner and the DNA writer incorporates DNA sequences in the DSB. The endogenous DNA repair machinery synthesizes repairs the DSB. As such, the combination of the DNA cutter and the DNA writer modifies the target DNA and leaves “marks” of the occurrence of the cellular event of interest. These marks are sequenced and the cellular event history of the cell is deciphered.