Apr 08, 2026
FACS Analysis of Mitochondrial Membrane Potential and CRISPR Screen Read-out via Amplicon Sequencing
- 1Yale University, USA;
- 2Mohn Research Centre for Regenerative Medicine, Norway
- Brennand Laboratory
Protocol Citation: Novin Balafkan, Kristen Brennand 2026. FACS Analysis of Mitochondrial Membrane Potential and CRISPR Screen Read-out via Amplicon Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyobwwgx9/v1
Manuscript citation:
Garcia, Meilin Fernandez, et al. "Dynamic convergence of neurodevelopmental disorder risk genes across neurodevelopment." bioRxiv (2025): 2024-08.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 07, 2026
Last Modified: April 08, 2026
Protocol Integer ID: 314671
Keywords: JC-1 staining, Mitochondrial membrane potential (MMP), FACS, CRISPR screen, Amplicon sequencing, facs analysis of mitochondrial membrane potential, mitochondrial membrane potential, affecting mitochondrial function, mitochondrial function, combining mitochondrial membrane potential, crispr screen read, assessment of gene perturbation, genomic dna, gene perturbation, rna abundance
Funders Acknowledgements:
European Union Horizon 2020 (MSCA)
Grant ID: 101065629
NIH National Institute of Mental Health
Grant ID: R01MH123155
Abstract
This protocol describes a two-part assay combining mitochondrial membrane potential (Δψm) measurement via JC-1 dye staining with CRISPR screen read-out through amplicon sequencing. Following differentiation of hPSCs to NPCs or iGlut neurons, cells are stained with JC-1 and analyzed by FACS to quantify Δψm shifts. Genomic DNA is extracted from sorted cells, and guide RNA abundance is quantified via UMI-based amplicon sequencing. This method enables assessment of gene perturbations affecting mitochondrial function at the single-guide resolution.
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