Pooled image-base screening of mitochondria with microraft isolation distinguishes pathogenic mitofusin 2 mutations

• Published in: Communications biology Vol. 5; no. 1; pp. 1128 - 14
• Main Authors: Yenkin, Alex L., Bramley, John C., Kremitzki, Colin L., Waligorski, Jason E., Liebeskind, Mariel J., Xu, Xinyuan E., Chandrasekaran, Vinay D., Vakaki, Maria A., Bachman, Graham W., Mitra, Robi D., Milbrandt, Jeffrey D., Buchser, William J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 25.10.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 13/109; 13/31; 14; 14/19; 14/34; 45/23; 45/70; 631/208/191; 631/208/191/1908; 631/80/2373; 631/80/642/333; Biology; Biomedical and Life Sciences; Gene Editing; Genetic diversity; Genetic screening; Genomics - methods; Humans; Learning algorithms; Life Sciences; Machine learning; Mitochondria; Mitochondria - genetics; Mutation; Phenotype; Phenotypes
• ISSN: 2399-3642; 2399-3642
• DOI: 10.1038/s42003-022-04089-y

Most human genetic variation is classified as variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a method termed “Raft-Seq”. We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator Mitofusin 2, even when the cellular phenotype is subtle. We also show that our platform achieves its efficacy using multiple cellular features, which can be configured on-the-fly. Raft-Seq enables a way to perform pooled screening on sets of mutations in biologically relevant cells, with the ability to physically capture any cell with a perturbed phenotype and expand it clonally, directly from the primary screen. Raft-Seq is a generalizable pooled screening platform that combines high-content imaging, machine learning and microraft isolation, and enables efficient screening of genetic perturbations based on their impact on phenotypes.