LipoGrid: A High-Throughput Multi-omics Perturbation Screen Dissects the Genetic Architecture of Lipid Metabolism

Lipids constitute one of the largest and most diverse classes of cellular molecules, sustaining membrane architecture, energy storage, and signaling. Consequently, their dysregulation underlies a broad spectrum of human disease. However, the genetic mechanisms governing lipid homeostasis have remained largely inaccessible, owing to the lack of approaches capable of systematically linking defined genetic perturbations to large-scale changes in cellular lipidome composition. Here we introduce LipoGrid, a spatial mass spectrometry platform that resolves the genetic architecture of lipid metabolism at single-cell resolution. LipoGrid arrays CRISPR/Cas9-perturbed cells on a micropatterned grid and sequentially captures lipidomic and gRNA identity from the same cells, complemented by single-cell RNA sequencing of matched cell populations subjected to the same perturbations. Using this approach, we quantified the relative abundance of 158 distinct lipid species across 143 target genes in a rigorously controlled experimental framework. We find that most gene knockouts produced measurable alterations in lipid composition, often affecting specific lipid classes and molecular subspecies. The screen accurately recapitulated established gene-lipid relationships, including enzyme-substrate specificities, lipid pathway regulators, and disease-associated loss-of-function phenotypes, thereby demonstrating the sensitivity and accuracy of LipoGrid. By jointly profiling transcriptomic and lipidomic responses, we further uncover compensatory feedback mechanisms that buffer the impact of genetic perturbations on the cellular lipidome. Collectively, these findings establish LipoGrid as a scalable multimodal platform for systematically mapping gene-lipid interactions and reveal the regulatory networks linking gene perturbation, transcriptional adaptation, and lipidome remodeling. HighlightsO_LIMicropatterned single-cell growth enables spatial lipidomic perturbation screens C_LIO_LILipoGrid maps 143 gene knockouts to 158 lipid species and transcriptomic states C_LIO_LIPerturbed lipidomes reveal compensatory feedback and lipid-class-specific uptake C_LIO_LIRecovers enzyme substrate specificities and disease-linked lipid signatures C_LI