In Vivo Gene Function Evaluation via Single-Cell Barcoding
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Solution Overview
Problem
Current methods for identifying the point of action of risk-associated genes in human diseases are limited, as each gene can affect multiple tissues, cell types, and molecular pathways, making high-resolution phenotyping and individual knockout animal models impractical for initial functional investigation.
Innovation Solution
The development of methods for in vivo evaluation of gene functions involves introducing genetic perturbations into progenitor cells in an animal model, coupled with a reporter gene and barcode, allowing for the profiling of genomic, genetic, epigenetic, proteomic, or phenotypic changes in progeny cells to reveal gene function in a cell type-specific manner.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If individual knockout animal models are generated for each risk-associated gene, then the function of each gene can be investigated, but the time and resource requirements become prohibitive
Solution Approach 1:
The patent segments the investigation process by introducing multiple genetic perturbations simultaneously into a single animal model rather than creating separate knockout models for each gene. This allows parallel evaluation of multiple genes through single-cell RNA sequencing, dramatically reducing the time required while maintaining the ability to identify specific gene functions and their cell type-specific effects
Solution Approach 2:
The patent creates a universal platform that can evaluate multiple risk-associated genes simultaneously using the same animal model and sequencing approach. This multi-functional system allows researchers to investigate the functions of many genes in parallel across different cell types without needing to develop separate models for each gene
2Measurement precision
If high-resolution phenotyping methods are used to identify tissue- and cell-type specific effects, then gene function can be precisely determined, but the complexity and cost of individual knockout models increases
Solution Approach 1:
The patent applies segmentation by using single-cell RNA sequencing to analyze gene expression at the individual cell level rather than averaging across entire tissues. This allows precise identification of which specific cell types are affected by each genetic perturbation, providing high-resolution phenotyping data without requiring complex multi-model approaches
Solution Approach 2:
The patent introduces barcode sequences as intermediaries that link specific genetic perturbations to their resulting cell populations. These barcodes allow researchers to track and identify the progeny of perturbed cells through sequencing, serving as a mediator that connects the genetic modification to the phenotypic outcome in a tractable way
3Productivity
If multiple genetic perturbations are introduced into progenitor cells in parallel, then gene functions can be evaluated efficiently, but the complexity of delivering and tracking multiple perturbations increases
Solution Approach 1:
The patent merges multiple genetic perturbations into a single viral delivery system that can introduce multiple barcoded perturbations simultaneously into progenitor cells. This combining approach allows efficient parallel evaluation of many genes while managing delivery complexity through a unified vector system that handles multiple genetic elements in one administration
Data Source
AI summary
Described herein are methods and uses thereof for in vivo evaluating functions of multiple genes in parallel by combining in utero genetic perturbation of progenitor cells and single-cell transcriptomic profiling of progeny cells in animals. These methods can be used, among other things, to reveal in vivo gene functions in a cell type-specific manner.