The Regents of the University of California (20240327823). METHODS FOR SCREENING GENETIC PERTURBATIONS simplified abstract
Contents
METHODS FOR SCREENING GENETIC PERTURBATIONS
Organization Name
The Regents of the University of California
Inventor(s)
Prashant Mali of La Jolla CA (US)
Udit Parekh of La Jolla CA (US)
METHODS FOR SCREENING GENETIC PERTURBATIONS - A simplified explanation of the abstract
This abstract first appeared for US patent application 20240327823 titled 'METHODS FOR SCREENING GENETIC PERTURBATIONS
Abstract: 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.
Key Features and Innovation:
- Use of barcoded ORF overexpression libraries with scRNA-seq and fitness screening for comprehensive analysis of genetic perturbations in hPSCs.
- Systematic assay of mutant gene libraries and whole gene families to identify key altered gene modules.
- Identification of specific transcription factors like KLF4 and SNAI2 with opposing effects on pluripotency gene module.
- Discovery of ETV2 as a driver of reprogramming towards an endothelial-like state.
Potential Applications:
- Understanding the effects of genetic perturbations on cellular state and fitness in hPSCs.
- Identifying key transcription factors and genes that influence pluripotency and reprogramming towards specific cell states.
Problems Solved:
- Overcoming the limitations of traditional pooled screening techniques for analyzing genetic perturbations in hPSCs.
- Providing a comprehensive platform for assessing the impact of transcription factors and genes on cellular fitness and transcriptomic profiles.
Benefits:
- Enhanced understanding of the complex effects of genetic perturbations on hPSCs.
- Identification of key regulators of pluripotency and reprogramming processes.
Commercial Applications: Title: Comprehensive Genetic Perturbation Analysis Platform for hPSCs Potential commercial uses include drug discovery, regenerative medicine research, and personalized medicine development. Market implications include improved efficiency in identifying key regulators of cellular state and fitness in hPSCs.
Prior Art: Readers can explore prior research on single-cell RNA sequencing, fitness screening techniques, and genetic perturbation analysis in hPSCs to understand the evolution of this technology.
Frequently Updated Research: Stay updated on the latest advancements in single-cell sequencing technologies, gene editing tools, and stem cell research to enhance the applications of this comprehensive genetic perturbation analysis platform.
Questions about Genetic Perturbation Analysis in hPSCs: 1. How does the SEUSS technique improve upon traditional pooled screening methods for analyzing genetic perturbations in hPSCs? 2. What are the potential implications of identifying key transcription factors and genes that influence pluripotency and reprogramming processes in hPSCs?
Original Abstract Submitted
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.
