2. Academic Validation
  3. Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells

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

  • bioRxiv. 2025 Jan 23:2025.01.21.633969. doi: 10.1101/2025.01.21.633969.
Sushama Sivakumar 1,  Yihan Wang 2,  Sean C Goetsch 1,  Vrushali Pandit 1,  Lei Wang 2,  Huan Zhao 2,  Anjana Sundarrajan 2,  Daniel Armendariz 2,  Chikara Takeuchi 2,  Mpathi Nzima 2,  Wei-Chen Chen 3,  Ashley E Dederich 4,  Lauretta El Hayek 3 5,  Taosha Gao 2,  Renad Ghazawi 4,  Ashlesha Gogate 3 5,  Kiran Kaur 3 5,  Hyung Bum Kim 2,  Melissa K McCoy 4,  Hanspeter Niederstrasser 4,  Seiya Oura 6,  Carolos A Pinzon-Arteaga 6,  Menaka Sanghvi 6,  Daniel A Schmitz 6,  Leqian Yu 6,  Yanfeng Zhang 7,  Qinbo Zhou 7,  W Lee Kraus 2,  Lin Xu 7 8,  Jun Wu 6,  Bruce A Posner 4,  Maria H Chahrour 3 5 9 10 11,  Gary C Hon 2 12,  Nikhil V Munshi 1 2 5 6 13
Affiliations

Affiliations

  • 1 Department of Internal Medicine, Division of Cardiology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 2 Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 3 Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 4 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 5 Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 6 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 7 Quantitative Biomedical Research Center, Peter O'Donnell Jr School of Public Health, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 8 Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 9 Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 10 Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 11 Peter O'Donnell Jr Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 12 Lyda Hill Department of Bioinformatics, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
  • 13 Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.
PMID: 39896670 DOI: 10.1101/2025.01.21.633969
Abstract

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 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.

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