Mitochondrial Phenotypes Distinguish Pathogenic MFN2 Mutations by Pooled Functional Genomics Screen

Most human genetic variation is classified as VUS - 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 new method termed "Raft-Seq". We highlight the efficacy of our platform by showing its ability to distinguish pathogenic point mutations of the mitochondrial regulator MFN2, 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 new 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/434746v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@c774b4org.highwire.dtl.DTLVardef@bee63corg.highwire.dtl.DTLVardef@fd7099org.highwire.dtl.DTLVardef@eb7d87_HPS_FORMAT_FIGEXP M_FIG C_FIG Here, we address the need to evaluate the impact of numerous genetic variants. This manuscript depicts the methods of using machine learning on a biologically relevant phenotype to predict specific point mutations, followed by physically capturing those mutated cells.