Fig 1. Arrayed transmembrane protein screening and pooled genome-wide CRISPR activation screening identify LRRC15 as binding SARS-CoV-2 spike protein.

(A) Schematic of arrayed cell-based screening to identify host factors that cause SARS-CoV-2 spike protein binding. HEK293 cells were transfected in individual wells of microtiter plates with full-length cDNA constructs encompassing a near-comprehensive library of human membrane proteins. Each well in the array was tested for binding to fluorescent tetramers of full-length SARS-CoV-2 spike by flow cytometry. (B) Schematic of pooled CRISPR activation screening. RPE1 cells expressing a SunTag CRISPRa system were transduced with guide RNAs to activate transcription of all genes in the human genome. Cells that bound to Fc protein fusions of the spike S1 domain were sorted by FACS and sequenced to measure guide RNA enrichment. (C) Top-ranked hits from arrayed cDNA screening. Distributions of cell fluorescence by flow cytometry are shown after incubation with fluorescent spike protein tetramers for cDNAs, which produced top-ranked signals in the arrayed screen. CD2 is included as a negative control. (D) Guide RNA enrichment of genes promoting SARS-CoV2 spike protein binding using CRISPR activation screening. Genes are ordered according to their positions across the genome and the statistical significance of their respective guide RNA enrichments post-sorting. (E) Independent transfections of LRRC15 cDNA induce spike binding. Flow cytometry traces for HEK293 cells transiently transfected with LRRC15 cDNA compared to mock-transfected controls. Different quantities of full-length spike protein were applied, as indicated along the y-axis. (F) Single-guide RNA clones validate LRRC15 as a gene that induces spike binding. Flow cytometry traces for RPE1 cells transduced and selected for individual LRRC15-activating guide RNAs. CRISPRa, CRISPR activation; LRRC15, leucine-rich repeat containing protein 15; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.