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Cell ; 184(1): 92-105.e16, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1064907


To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

COVID-19/genetics , COVID-19/virology , Host-Pathogen Interactions , SARS-CoV-2/physiology , A549 Cells , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/virology , Angiotensin-Converting Enzyme 2/metabolism , Biosynthetic Pathways , COVID-19/metabolism , Cholesterol/biosynthesis , Clustered Regularly Interspaced Short Palindromic Repeats , Endosomes/metabolism , Gene Expression Profiling , Gene Knockdown Techniques , Gene Knockout Techniques/methods , Genome-Wide Association Study , Host-Pathogen Interactions/drug effects , Humans , RNA Interference , SARS-CoV-2/growth & development , Single-Cell Analysis , Viral Load/drug effects , rab GTP-Binding Proteins/genetics
Nucleic Acids Res ; 49(7): e40, 2021 04 19.
Article in English | MEDLINE | ID: covidwho-1050155


Generation of conditional knockout (cKO) and various gene-modified cells is laborious and time-consuming. Here, we established an all-in-one cKO system, which enables highly efficient generation of cKO cells and simultaneous gene modifications, including epitope tagging and reporter gene knock-in. We applied this system to mouse embryonic stem cells (ESCs) and generated RNA helicase Ddx1 cKO ESCs. The targeted cells displayed endogenous promoter-driven EGFP and FLAG-tagged DDX1 expression, and they were converted to Ddx1 KO via FLP recombinase. We further established TetFE ESCs, which carried a reverse tetracycline transactivator (rtTA) expression cassette and a tetracycline response element (TRE)-regulated FLPERT2 cassette in the Gt(ROSA26)Sor locus for instant and tightly regulated induction of gene KO. By utilizing TetFE Ddx1F/F ESCs, we isolated highly pure Ddx1F/F and Ddx1-/- ESCs and found that loss of Ddx1 caused rRNA processing defects, thereby activating the ribosome stress-p53 pathway. We also demonstrated cKO of various genes in ESCs and homologous recombination-non-proficient human HT1080 cells. The frequency of cKO clones was remarkably high for both cell types and reached up to 96% when EGFP-positive clones were analyzed. This all-in-one cKO system will be a powerful tool for rapid and precise analyses of gene functions.

DEAD-box RNA Helicases/metabolism , Gene Knockout Techniques/methods , RNA, Ribosomal/metabolism , Animals , Cell Line , Embryonic Stem Cells , Fibroblasts , Gene Expression , Gene Knock-In Techniques , Humans , Mice , Mice, Inbred C57BL , RNA Processing, Post-Transcriptional , Ribosomes/metabolism