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

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.

Automated summary

In this paper, the authors present a functional genomics platform called Raft-Seq, which combines high-content imaging, machine learning, and microraft isolation to identify cellular phenotypes underlying human diseases. The platform demonstrates its efficacy in distinguishing pathogenic mutations, even when the cellular phenotype is subtle, and utilizes multiple cellular features that can be configured on-the-fly.