Genetic Scratchpad Lineage Tracking via CRISPR
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Solution Overview
Problem
Current methods for determining cell lineages in complex organisms are limited, as they cannot systematically record molecular events or reconstruct entire lineage trees, and often require significant perturbation to cells and organisms.
Innovation Solution
A method and system that introduce molecular changes into genetic scratchpads in cells over multiple cell cycle generations, allowing for the characterization of molecular changes and establishment of lineage connections by comparing mutation statuses using probes and guide molecules like CRISPR-Cas9, enabling the recording and reading out of lineage information within intact cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If site-specific recombinases (FLP, Cre) are used to mark cell descendants, then lineage tracking capability is improved, but the ability to follow multiple lineage decisions and reconstruct entire lineage trees is limited
Solution Approach 1:
The invention divides the genome into multiple independent genetic scratchpads, each capable of recording specific molecular events. By segmenting the recording function across multiple scratchpads, the system can simultaneously track multiple lineage decisions and molecular events, overcoming the limitation of single-marker approaches like FLP or Cre recombinases.
Solution Approach 2:
The genetic scratchpad system serves multiple functions: it records molecular events, tracks cell lineage, and enables reconstruction of entire lineage trees. The universal applicability of the scratchpad approach across different molecular events and cell types provides the versatility needed to follow multiple lineage decisions simultaneously.
2Loss of information
If existing lineage determination techniques are applied, then some lineage information can be obtained, but systematic recording of molecular events during lineage determination within cells themselves is not enabled
Solution Approach 1:
The genetic scratchpads are integrated directly into the cell's own genome, allowing cells to self-record their molecular events and lineage information endogenously. This self-service approach eliminates the need for external recording systems that would require extracting and analyzing cells, thereby minimizing perturbation while enabling systematic recording of molecular events.
3Measurement precision
If molecular changes are introduced to track lineage information, then lineage reconstruction capability is improved, but the complexity of the system increases
Solution Approach 1:
The invention uses uniform genetic scratchpad structures integrated throughout the genome, each with the same basic architecture for recording molecular events. This homogeneity in design simplifies the system compared to using multiple different types of markers or complex external recording systems, while still enabling comprehensive lineage reconstruction.
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 systematic tracking of lineage information and molecular events with minimal perturbation, allowing for the reconstruction of comprehensive lineage trees and the recording of cellular histories, providing detailed insights into developmental processes and tissue formation.
Implementation Method 1
guide molecules like CRISPR-Cas9
Implementation Method 2
each probe recognizes and binds to a corresponding target sequence in a target site
Data Source
AI summary
Methods and systems for recording and mapping lineage information and molecular events in individual cells are provided. Molecular changes, which may result from random or specific molecular events, are introduced to defined regions in cells over multiple cell cycle generations. Techniques such as fluorescent imaging are applied to track and identify the molecular changes before such information is used for lineage analysis or for identifying key processes and key players in cellular pathways.