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

Sushama Sivakumar¹, Yihan Wang², Sean C Goetsch¹, Vrushali Pandit¹, Lei Wang², Huan Zhao², Anjana Sundarrajan², Daniel Armendariz², Chikara Takeuchi², Minnie Deng¹, Mpathi Nzima², Wei-Chen Chen³, Ashley E Dederich⁴, Lauretta El Hayek⁵, Taosha Gao², Ashlesha Gogate⁵, Kiran Kaur⁵, Hyung Bum Kim², Melissa K McCoy⁴, Hanspeter Niederstrasser⁴, Seiya Oura⁶, Carlos A Pinzon-Arteaga⁶, Menaka Sanghvi⁶, Daniel A Schmitz⁶, Leqian Yu⁶, Yanfeng Zhang⁷, Qinbo Zhou⁷, W Lee Kraus², Lin Xu⁸, Jun Wu⁶, Bruce A Posner⁴, Maria H Chahrour⁹, Gary C Hon¹⁰, Nikhil V Munshi¹¹

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 Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA; 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 Quantitative Biomedical Research Center, Peter O'Donnell Jr School of Public Health, University of Texas Southwestern Medical Center, Dallas, TX, USA; Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁹ Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA; Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX, USA; Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX, USA; Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX, USA; Peter O'Donnell Jr Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.
¹⁰ Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA; Lyda Hill Department of Bioinformatics, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA. Electronic address: gary.hon@utsouthwestern.edu.
¹¹ Department of Internal Medicine, Division of Cardiology, University of Texas Southwestern Medical Center, Dallas, TX, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA; Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX, USA; Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA. Electronic address: nikhil.munshi@utsouthwestern.edu.

PMID: 41237780
DOI: 10.1016/j.stemcr.2025.102713

Free article

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

Sushama Sivakumar et al. Stem Cell Reports. 2025.

Free article

. 2025 Nov 13:102713.

doi: 10.1016/j.stemcr.2025.102713. Online ahead of print.

Authors

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 Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA; 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 Quantitative Biomedical Research Center, Peter O'Donnell Jr School of Public Health, University of Texas Southwestern Medical Center, Dallas, TX, USA; Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
⁹ Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, USA; Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX, USA; Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX, USA; Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, TX, USA; Peter O'Donnell Jr Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.
¹⁰ Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA; Lyda Hill Department of Bioinformatics, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA. Electronic address: gary.hon@utsouthwestern.edu.
¹¹ Department of Internal Medicine, Division of Cardiology, University of Texas Southwestern Medical Center, Dallas, TX, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA; Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX, USA; Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA. Electronic address: nikhil.munshi@utsouthwestern.edu.

PMID: 41237780
DOI: 10.1016/j.stemcr.2025.102713

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 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.

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

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Update of

Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells.
Sivakumar S, Wang Y, Goetsch SC, Pandit V, Wang L, Zhao H, Sundarrajan A, Armendariz D, Takeuchi C, Nzima M, Chen WC, Dederich AE, El Hayek L, Gao T, Ghazawi R, Gogate A, Kaur K, Kim HB, McCoy MK, Niederstrasser H, Oura S, Pinzon-Arteaga CA, Sanghvi M, Schmitz DA, Yu L, Zhang Y, Zhou Q, Kraus WL, Xu L, Wu J, Posner BA, Chahrour MH, Hon GC, Munshi NV. Sivakumar S, et al. bioRxiv [Preprint]. 2025 Jan 23:2025.01.21.633969. doi: 10.1101/2025.01.21.633969. bioRxiv. 2025. Update in: Stem Cell Reports. 2025 Nov 13:102713. doi: 10.1016/j.stemcr.2025.102713. PMID: 39896670 Free PMC article. Updated. Preprint.

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

Affiliations

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

Authors

Affiliations

Abstract

Conflict of interest statement

Update of

LinkOut - more resources

Full Text Sources