High-Throughput ORF Screening for Transcription Factor Identification
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Solution Overview
Problem
Current methods for cellular reprogramming lack a scalable screening approach to assess the effects of transcription factor overexpression, hindering the discovery of novel reprogramming factors and understanding of the differentiation process.
Innovation Solution
A high-throughput platform utilizing a novel open reading frame (ORF) gene overexpression vector library of developmentally critical transcription factors to build genetic co-perturbation networks and identify key reprogramming drivers through single-cell RNA sequencing, specifically identifying the role of ETV2 in endothelial differentiation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional trial-and-error methods are used to identify transcription factors for cellular reprogramming, then the process can be performed with existing knowledge and simple experimental setups, but the screening process is not scalable and time-consuming
Solution Approach 1:
The patent segments the screening process into modular components: a library of individual transcription factor expression vectors, a standardized reporter cell line system, and high-throughput transfection protocols. This segmentation enables parallel processing of multiple transcription factors simultaneously, transforming the traditional sequential trial-and-error approach into a scalable high-throughput screening platform that can evaluate numerous factors in a single experiment
Solution Approach 2:
The patent changes the experimental parameters by using a standardized reporter cell line with a constitutive promoter driving reporter gene expression. This parameter change allows for quantitative measurement of transcriptional activity across multiple cell types and conditions, enabling systematic comparison and identification of reprogramming factors without repeating complex experimental setups for each factor
2Loss of information
If comprehensive screening of transcription factors is performed to discover novel reprogramming factors, then the understanding of differentiation processes is improved, but the complexity of the screening platform increases
Solution Approach 1:
The patent creates a universal screening platform where a single reporter cell line design can be used across multiple cell types, transcription factors, and differentiation conditions. The standardized reporter construct with constitutive promoter and reporter gene serves multiple functions: measuring transcriptional activity, enabling quantitative comparison, and working across different biological systems. This universality reduces platform complexity by eliminating the need for separate experimental systems for each screening scenario
Solution Approach 2:
The patent introduces a standardized reporter cell line as an intermediary between the transcription factor expression vectors and the readout system. This intermediary layer simplifies the connection between diverse transcription factors and the measurement system, providing a consistent interface that translates various transcriptional activities into comparable quantitative data without requiring complex cell-type-specific assays
Data Source
AI summary
Understanding the complex effects of genetic perturbations on cellular state and fitness in human pluripotent stem cells (hPSCs) has been challenging using traditional pooled screening techniques which typically rely on unidimensional phenotypic readouts. Here, Applicants use barcoded open reading frame (ORF) overexpression libraries with a coupled single-cell RNA sequencing (scRNA-seq) and fitness screening approach, a technique we call SEUSS (ScalablE fUnctional Screening by Sequencing), to establish a comprehensive assaying platform. Using this system, Applicants perturbed hPSCs with a library of developmentally critical transcription factors (TFs), and assayed the impact of TF overexpression on fitness and transcriptomic cell state across multiple media conditions. Applicants further leveraged the versatility of the ORF library approach to systematically assay mutant gene libraries and also whole gene families. From the transcriptomic responses, Applicants built genetic co-perturbation networks to identify key altered gene modules. Strikingly, we found that KLF4 and SNAI2 have opposing effects on the pluripotency gene module, highlighting the power of this method to characterize the effects of genetic perturbations. From the fitness responses, Applicants identified ETV2 as a driver of reprogramming towards an endothelial-like state.