Fig. 1: Genome-wide CRISPR knockout screen identifies UHRF1 as a host restriction factor for HCoV-229E infection.

A. Identification of host restriction genes from the CRISPR knockout screen. The A549 cell library containing genome-wide CRISPR knockout sgRNAs were infected with HCoV-229E-mGreen (MOI 0.5, 24 h). Infected reporter-positive cells were sorted for genomic DNA extraction and subsequent sgRNA sequence analysis. Genes were analyzed using MAGeCK software and ranked based on -log10 (MAGeCK score) and P-values. The algorithm employs one-sided test to identify genes under positive selection, and P-values were adjusted for multiple testing using the Benjamini-Hochberg method. B Validation of the 12 top-ranked genes, with a cutoff of false discovery rate (FDR) < 0.05. A549 cells were edited with two independent sgRNAs per gene. Following infection with HCoV-229E (MOI 0.5, 24 h), infection efficiency was quantified by flow cytometry for the percentage of nucleocapsid (N)-positive cells. C Representative images of control and UHRF1-knockout A549 cells infected with HCoV-229E (MOI 0.5, 24 h), and analyzed by Operetta high-content imaging from three independent experiments. Scale bar, 100 μm. D Cell viability of control and gene-knockout A549 cells was assessed 48 h after seeding. E Overexpression of human UHRF1 inhibits HCoV-229E infection. HeLa cells overexpressing UHRF1 were infected with HCoV-229E (MOI 0.5, 24 h). Virus infection efficiency was determined by flow cytometry. F Trans-complementation of UHRF1 in knockout cells inhibits HCoV-229E infection. A549 cells with or without exogenous UHRF1 expression were edited with sgRNA targeting endogenous UHRF1, and infected with HCoV-229E (MOI 0.5, 24 h). Error bars represent standard deviations from three independent experiments (n = 3), and each performed in duplicate. Two-way ANOVA with Sidak’s test (B, F); unpaired, two-sided t-test (D, E); mean ± s.d.; *P < 0.05; ***P < 0.001; ****P < 0.0001; ns not significant.