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

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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