Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells
Perturb-seq is a powerful approach to systematically assess how genes and enhancers impact the molecular and cellular pathways of development and disease. However, technical challenges have limited its application in stem-cell-based systems. Here, we benchmarked Perturb-seq across multiple CRISPRi modalities, on diverse genomic targets, in multiple human pluripotent stem cells, during directed differentiation to multiple lineages, and across multiple single guide RNA (sgRNA) delivery systems. To ensure cost-effective production of large-scale Perturb-seq datasets as part of the Impact of Genomic Variants on Function (IGVF) consortium, our optimized protocol dynamically assesses experiment quality across the weeks-long procedure. Our analysis of 1,996,260 sequenced cells across benchmarking datasets reveals shared regulatory networks linking disease-associated enhancers and genes with downstream targets during cardiomyocyte differentiation. This study establishes open tools and resources for interrogating genome function during stem cell differentiation.
Authors
- Kiran Kaur
- Sushama Sivakumar (ORCID: https://orcid.org/0000-0001-7877-4821)
- Anjana Sundarrajan
- Daniel Armendariz (ORCID: https://orcid.org/0000-0002-5857-1282)
- Hanspeter Niederstrasser
- Qinbo Zhou (ORCID: https://orcid.org/0000-0002-6563-7964)
- Minnie Deng
- Vrushali Pandit (ORCID: https://orcid.org/0000-0002-0642-6438)
- Daniel A. Schmitz (ORCID: https://orcid.org/0000-0002-2112-5561)
- W. Lee Kraus (ORCID: https://orcid.org/0000-0002-8786-2986)
- Maria H. Chahrour (ORCID: https://orcid.org/0000-0002-3174-1480)
- Bruce A. Posner (ORCID: https://orcid.org/0000-0002-7033-4933)
- Sean C. Goetsch (ORCID: https://orcid.org/0000-0003-1141-683X)
- Lauretta El Hayek (ORCID: https://orcid.org/0000-0002-6820-9316)
- Melissa K. McCoy (ORCID: https://orcid.org/0009-0008-2186-9158)
- Hyung Bum Kim
- Seiya Oura (ORCID: https://orcid.org/0000-0002-7606-732X)
- Jun Wu (ORCID: https://orcid.org/0000-0002-6346-4756)
- Chikara Takeuchi (ORCID: https://orcid.org/0000-0001-6855-6580)
- Carlos A. Pinzón-Arteaga (ORCID: https://orcid.org/0000-0002-4906-6751)
- Wei-Chen Chen (ORCID: https://orcid.org/0000-0002-3711-818X)
- Lin Xu (ORCID: https://orcid.org/0000-0002-0537-922X)
- Ashlesha Gogate (ORCID: https://orcid.org/0009-0002-6081-1289)
- Yihan Wang (ORCID: https://orcid.org/0000-0002-1163-4610)
- Leqian Yu
- Taosha Gao
- Nikhil Munshi (ORCID: https://orcid.org/0000-0002-8397-100X)
- Gary C. Hon (ORCID: https://orcid.org/0000-0002-5728-4433)
- Ashley E Dederich
- Mpathi Nzima
- Huan Zhao
- Yanfeng Zhang
- Lei Wang (ORCID: https://orcid.org/0000-0003-0734-7026)
- Menaka Sanghvi
Institutions
- Southwestern Medical Center (US)
- The University of Texas Southwestern Medical Center (US)
Publication Details
- Journal
- Stem Cell Reports
- Published
- 2025-11-13
- DOI
- https://doi.org/10.1016/j.stemcr.2025.102713
- Citations
- 1
- Primary Topic
- Pluripotent Stem Cells Research
- Type
- article
- Field-Weighted Citation Impact
- 0.81