tetano
Editor, Senior Moderator
Nat Biomed Eng
. 2023 Nov 23.
doi: 10.1038/s41551-023-01140-z. Online ahead of print. High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2
Charles W F Chan[SUP] #[/SUP][SUP] 1 2 [/SUP], Bei Wang[SUP] #[/SUP][SUP] 1 2 [/SUP], Lang Nan[SUP] #[/SUP][SUP] 3 4 [/SUP], Xiner Huang[SUP] #[/SUP][SUP] 5 [/SUP], Tianjiao Mao[SUP] 3 4 [/SUP], Hoi Yee Chu[SUP] 1 2 [/SUP], Cuiting Luo[SUP] 5 [/SUP], Hin Chu[SUP] 6 7 8 [/SUP], Gigi C G Choi[SUP] 9 10 11 [/SUP], Anderson H C Shum[SUP] 12 13 [/SUP], Alan S L Wong[SUP] 14 15 [/SUP]
Affiliations
Mapping mutations and discovering cellular determinants that cause the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce infected cells to form syncytia would facilitate the development of strategies for blocking the formation of such cell-cell fusion. Here we describe high-throughput screening methods based on droplet microfluidics and the size-exclusion selection of syncytia, coupled with large-scale mutagenesis and genome-wide knockout screening via clustered regularly interspaced short palindromic repeats (CRISPR), for the large-scale identification of determinants of cell-cell fusion. We used the methods to perform deep mutational scans in spike-presenting cells to pinpoint mutable syncytium-enhancing substitutions in two regions of the spike protein (the fusion peptide proximal region and the furin-cleavage site). We also used a genome-wide CRISPR screen in cells expressing the receptor angiotensin-converting enzyme 2 to identify inhibitors of clathrin-mediated endocytosis that impede syncytium formation, which we validated in hamsters infected with SARS-CoV-2. Finding genetic and cellular determinants of the formation of syncytia may reveal insights into the physiological and pathological consequences of cell-cell fusion.
. 2023 Nov 23.
doi: 10.1038/s41551-023-01140-z. Online ahead of print. High-throughput screening of genetic and cellular drivers of syncytium formation induced by the spike protein of SARS-CoV-2
Charles W F Chan[SUP] #[/SUP][SUP] 1 2 [/SUP], Bei Wang[SUP] #[/SUP][SUP] 1 2 [/SUP], Lang Nan[SUP] #[/SUP][SUP] 3 4 [/SUP], Xiner Huang[SUP] #[/SUP][SUP] 5 [/SUP], Tianjiao Mao[SUP] 3 4 [/SUP], Hoi Yee Chu[SUP] 1 2 [/SUP], Cuiting Luo[SUP] 5 [/SUP], Hin Chu[SUP] 6 7 8 [/SUP], Gigi C G Choi[SUP] 9 10 11 [/SUP], Anderson H C Shum[SUP] 12 13 [/SUP], Alan S L Wong[SUP] 14 15 [/SUP]
Affiliations
- PMID: 37996617
- DOI: 10.1038/s41551-023-01140-z
Mapping mutations and discovering cellular determinants that cause the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce infected cells to form syncytia would facilitate the development of strategies for blocking the formation of such cell-cell fusion. Here we describe high-throughput screening methods based on droplet microfluidics and the size-exclusion selection of syncytia, coupled with large-scale mutagenesis and genome-wide knockout screening via clustered regularly interspaced short palindromic repeats (CRISPR), for the large-scale identification of determinants of cell-cell fusion. We used the methods to perform deep mutational scans in spike-presenting cells to pinpoint mutable syncytium-enhancing substitutions in two regions of the spike protein (the fusion peptide proximal region and the furin-cleavage site). We also used a genome-wide CRISPR screen in cells expressing the receptor angiotensin-converting enzyme 2 to identify inhibitors of clathrin-mediated endocytosis that impede syncytium formation, which we validated in hamsters infected with SARS-CoV-2. Finding genetic and cellular determinants of the formation of syncytia may reveal insights into the physiological and pathological consequences of cell-cell fusion.