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Updated: May 12, 2026

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Published on: May 29, 2014

Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells.

Sushama Sivakumar¹, Yihan Wang², Sean C Goetsch¹

¹Department of Internal Medicine, Division of Cardiology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Stem Cell Reports

|November 14, 2025

Summary

This summary is machine-generated.

Perturb-seq systematically assesses gene and enhancer functions in human stem cells. This study optimizes Perturb-seq for large-scale studies, revealing shared regulatory networks in cardiomyocyte differentiation.

Keywords:

CRISPR/Cas9 Perturb-seq cardiomyocytes human pluripotent stem cells neural progenitor cells single-cell genomics

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Area of Science:

Genomics
Stem Cell Biology
Molecular Biology

Background:

Perturb-seq is valuable for studying gene and enhancer functions in development and disease.
Technical hurdles have previously limited its use in stem cell systems.

Purpose of the Study:

To benchmark Perturb-seq across various CRISPR interference (CRISPRi) methods, genomic targets, and human pluripotent stem cells.
To optimize Perturb-seq for cost-effective, large-scale data generation within the Impact of Genomic Variants on Function (IGVF) consortium.
To establish open tools and resources for investigating genome function in stem cell differentiation.

Main Methods:

Benchmarking Perturb-seq using multiple CRISPRi modalities and single guide RNA (sgRNA) delivery systems.
Applying the optimized protocol to diverse human pluripotent stem cells undergoing directed differentiation.
Implementing dynamic quality assessment for week-long experiments.
Analyzing data from 1,996,260 sequenced cells.

Main Results:

Identified shared regulatory networks connecting disease-associated enhancers and genes during cardiomyocyte differentiation.
Demonstrated the robustness and scalability of the optimized Perturb-seq protocol.
Provided a comprehensive analysis of genome function interrogation in stem cell differentiation.

Conclusions:

The optimized Perturb-seq protocol overcomes previous technical limitations for stem cell applications.
This work provides valuable insights into regulatory networks governing cell fate decisions.
Open tools and resources are now available for future genome function studies.