Highlights d Lung ACE2 levels do not vary by age or sex, but smokers exhibit upregulated ACE2 d ACE2 is expressed in several lung cell types, including the secretory lineage d Chronic smoking triggers the expansion of ACE2 + secretory cells d ACE2 is also upregulated by viral infections and interferon exposure

The coronavirus SARS-CoV-2 has infected more than 600,000 people and has overwhelmed hospital systems around the world. However, the factors mediating fatal SARS-CoV-2 infections are poorly understood. Here, we show that cigarette smoke causes a dose-dependent upregulation of Angiotensin Converting Enzyme 2 (ACE2), the SARS-CoV-2 receptor, in rodent and human lungs. Using single-cell sequencing data, we demonstrate that ACE2 is expressed in a subset of epithelial cells that line the respiratory tract, including goblet cells, club cells, and alveolar type 2 cells. Chronic smoke exposure triggers a protective expansion of mucus-secreting goblet cells and a concomitant increase in ACE2 expression. In contrast, quitting smoking causes a decrease in lung ACE2 levels. Taken together, these results may partially explain why smokers are particularly likely to develop severe SARS-CoV-2 infections, and they suggest that quitting smoking could lessen coronavirus susceptibility.

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CRISPR/Cas9 technology enables the rapid generation of loss‐of‐function mutations in a targeted gene in mammalian cells. A single cell harboring those mutations can be used to establish a new cell line, thereby creating a CRISPR‐induced knockout clone. These clonal cell lines serve as crucial tools for exploring protein function, analyzing the consequences of gene loss, and investigating the specificity of biological reagents. However, the successful derivation of knockout clones can be technically challenging and may be complicated by multiple factors, including incomplete target ablation and interclonal heterogeneity. Here, we describe optimized protocols and plasmids for generating clonal knockouts in mammalian cell lines. We provide strategies for guide RNA design, CRISPR delivery, and knockout validation that facilitate the derivation of true knockout clones and are amenable to multiplexed gene targeting. These protocols will be broadly useful for researchers seeking to apply CRISPR to study gene function in mammalian cells. © 2019 The Authors.

Most cancers exhibit aneuploidy, but its functional significance in tumor development is controversial. Here, we describe ReDACT ( Re storing D isomy in A neuploid cells using C RISPR T argeting), a set of chromosome engineering tools that allow us to eliminate specific aneuploidies from cancer genomes. Using ReDACT, we created a panel of isogenic cells that have or lack common aneuploidies, and we demonstrate that trisomy of chromosome 1q is required for malignant growth in cancers harboring this alteration. Mechanistically, gaining chromosome 1q increases the expression of MDM4 and suppresses p53 signaling, and we show that TP53 mutations are mutually-exclusive with 1q aneuploidy in human cancers. Thus, tumor cells can be dependent on specific aneuploidies, raising the possibility that these “aneuploidy addictions” could be targeted as a therapeutic strategy.

Most cancers exhibit aneuploidy, but its functional significance in tumor development is controversial. Here, we describe ReDACT (Restoring Disomy in Aneuploid cells using CRISPR Targeting), a set of chromosome engineering tools that allow us to eliminate specific aneuploidies from cancer genomes. Using ReDACT, we created a panel of isogenic cells that have or lack common aneuploidies, and we demonstrate that trisomy of chromosome 1q is required for malignant growth in cancers harboring this alteration. Mechanistically, gaining chromosome 1q increases the expression of MDM4 and suppresses TP53 signaling, and we show that TP53 mutations are mutually-exclusive with 1q aneuploidy in human cancers. Thus, specific aneuploidies play essential roles in tumorigenesis, raising the possibility that targeting these "aneuploidy addictions" could represent a novel approach for cancer treatment.

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