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  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the recently emerged virus responsible for the COVID-19 pandemic. Clinical presentation can range from asymptomatic disease and mild respiratory tract infection to severe disease with lung injury, multiorgan failure, and death. SARS-CoV-2 is the third animal coronavirus to emerge in humans in the 21st century, and coronaviruses appear to possess a unique ability to cross borders between species and infect a wide range of organisms. This is somewhat surprising as, except for the requirement of host cell receptors, cell–pathogen interactions are usually species-specific. Insights into these host–virus interactions will provide a deeper understanding of the process of SARS-CoV-2 infection and provide a means for the design and development of antiviral agents. In this study, we describe a complex analysis of SARS-CoV-2 infection using a genome-wide CRISPR-Cas9 knock-out system in HeLa cells overexpressing entry receptor angiotensin-converting enzyme 2 (ACE2). This platform allows for the identification of factors required for viral replication. This study was designed to include a high number of replicates (48 replicates; 16 biological repeats with 3 technical replicates each) to prevent data instability, remove sources of bias, and allow multifactorial bioinformatic analyses in order to study the resulting interaction network. The results obtained provide an interesting insight into the replication mechanisms of SARS-CoV-2.

 嚴(yán)重急性呼吸系統(tǒng)綜合癥冠狀病毒 2 (SARS-CoV-2) 是最近出現(xiàn)的導(dǎo)致 COVID-19 大流行的病毒。臨床表現(xiàn)的范圍可以從無癥狀疾病和輕度呼吸道感染到嚴(yán)重的肺損傷、多器官衰竭和死亡。SARS-CoV-2 是 21 世紀(jì)第三種在人類中出現(xiàn)的動(dòng)物冠狀病毒，冠狀病毒似乎具有獨(dú)特的跨越物種邊界并感染廣泛生物體的能力。這有點(diǎn)令人驚訝，因?yàn)槌诵枰拗骷?xì)胞受體外，細(xì)胞-病原體相互作用通常是物種特異性的。對(duì)這些宿主-病毒相互作用的深入了解將有助于更深入地了解 SARS-CoV-2 感染的過程，并為設(shè)計(jì)和開發(fā)抗病毒藥物提供一種手段。在這項(xiàng)研究中，我們描述了在過度表達(dá)進(jìn)入受體血管緊張素轉(zhuǎn)換酶 2 (ACE2) 的 HeLa 細(xì)胞中使用全基因組 CRISPR-Cas9 敲除系統(tǒng)對(duì) SARS-CoV-2 感染進(jìn)行的復(fù)雜分析。該平臺(tái)允許識(shí)別病毒復(fù)制所需的因素。本研究旨在包括大量重復(fù)（48 個(gè)重復(fù)；16 個(gè)生物學(xué)重復(fù)，每個(gè)重復(fù) 3 個(gè)技術(shù)重復(fù)），以防止數(shù)據(jù)不穩(wěn)定，消除偏差來源，并允許進(jìn)行多因素生物信息學(xué)分析，以研究由此產(chǎn)生的相互作用網(wǎng)絡(luò)。獲得的結(jié)果提供了對(duì) SARS-CoV-2 復(fù)制機(jī)制的有趣見解。該平臺(tái)允許識(shí)別病毒復(fù)制所需的因素。本研究旨在包括大量重復(fù)（48 個(gè)重復(fù)；16 個(gè)生物學(xué)重復(fù)，每個(gè)重復(fù) 3 個(gè)技術(shù)重復(fù)），以防止數(shù)據(jù)不穩(wěn)定，消除偏差來源，并允許進(jìn)行多因素生物信息學(xué)分析，以研究由此產(chǎn)生的相互作用網(wǎng)絡(luò)。獲得的結(jié)果提供了對(duì) SARS-CoV-2 復(fù)制機(jī)制的有趣見解。該平臺(tái)允許識(shí)別病毒復(fù)制所需的因素。本研究旨在包括大量重復(fù)（48 個(gè)重復(fù)；16 個(gè)生物學(xué)重復(fù)，每個(gè)重復(fù) 3 個(gè)技術(shù)重復(fù)），以防止數(shù)據(jù)不穩(wěn)定，消除偏差來源，并允許進(jìn)行多因素生物信息學(xué)分析，以研究由此產(chǎn)生的相互作用網(wǎng)絡(luò)。獲得的結(jié)果提供了對(duì) SARS-CoV-2 復(fù)制機(jī)制的有趣見解。

 
高效搖瓶5L

Further knock-outs, including MACF1, PCDHGA1, GAGE1, and SPATA25, resulted in a decrease of viral RNA copies compared to non-targeting control (NTC). Microtubule-actin crosslinking factor 1 (MACF1), also widely known as actin crosslinking factor 7 (ACF7), plays a role in various cellular processes, including the regulation of cell polarization and motility through an interaction with microtubules and F-actin. In the context of coronavirus infection, microtubules and actin are critical in the transportation of internalized virus-containing vesicles. Interestingly, ACF7 is also involved in a wide range of cellular signaling networks, including Wnt/β-catenin signaling, the upregulation of which is associated with inflammation and cytokine storm in COVID-19 patients [46,47]. SPATA25 (spermatogenesis-associated protein 25) may play a role in spermatogenesis; however, this role is poorly understood. GAGE1 is a poorly defined protein, and its role in SARS-CoV-2 infection is currently unknown. Further examination of our top 178 list of targets and literature, suggested two further genes for validation: B4GALT7 and IL10RB.

B4GALT7 is a galactosyltransferase that functions in the heparan sulfate biosynthesis pathway and plays a role in DENV viral replication. B4GALT7 was identified in our screen and others [11,12]. The IL10 subunit IL10RB was similarly identified from our top 178 list of targets. Protein levels of IL10 may influence COVID-19 disease outcomes [48]. We, therefore, tested whether the depletion of these genes has an impact on SARS-CoV-2 replication, and using RT-qPCR analysis we found that both B4GALT7 and IL10RB KOs significantly reduced viral replication (Supplementary Figure S1), suggesting that they may play a role in SARS-CoV-2 infection. However, one may remember that using this methodology, only factors preventing productive infection would be identified.

細(xì)胞轉(zhuǎn)瓶5L

We have prepared an extensive dataset using our genome-wide CRISPR/Cas9 knock-out screening approach, resulting in the identification of several cellular factors required for SARS-CoV-2 replication. This study was highly repetitive to remove false positives and stochastic noise, as the experiment design is very complex. We have identified and validated select factors using a broad range of analytic and experimental approaches. The data described provide an interesting insight into SARS-CoV-2 viral replication mechanisms and identify factors that may serve as potential therapeutic targets.

 包括MACF1、PCDHGA1、GAGE1和SPATA25，與非靶向?qū)φ?(NTC) 相比，導(dǎo)致病毒 RNA 拷貝減少。微管-肌動(dòng)蛋白交聯(lián)因子 1 (MACF1)，也廣泛稱為肌動(dòng)蛋白交聯(lián)因子 7 (ACF7)，在各種細(xì)胞過程中發(fā)揮作用，包括通過與微管和 F-肌動(dòng)蛋白的相互作用調(diào)節(jié)細(xì)胞極化和運(yùn)動(dòng)。在冠狀病毒感染的背景下，微管和肌動(dòng)蛋白對(duì)于內(nèi)化含有病毒的囊泡的運(yùn)輸至關(guān)重要。有趣的是，ACF7 還涉及廣泛的細(xì)胞信號(hào)網(wǎng)絡(luò)，包括 Wnt/β-catenin 信號(hào)，其上調(diào)與 COVID-19 患者的炎癥和細(xì)胞因子風(fēng)暴有關(guān) [ 46 , 47]。SPATA25（精子發(fā)生相關(guān)蛋白25）可能在精子發(fā)生中起作用；然而，人們對(duì)這種作用知之甚少。GAGE1 是一種定義不清的蛋白質(zhì)，目前尚不清楚其在 SARS-CoV-2 感染中的作用。進(jìn)一步檢查我們的前 178 個(gè)目標(biāo)和文獻(xiàn)列表，建議進(jìn)一步驗(yàn)證兩個(gè)基因：B4GALT7 和 IL10RB。

10層細(xì)胞工廠

B4GALT7 是一種半乳糖基轉(zhuǎn)移酶，在硫酸乙酰肝素生物合成途徑中發(fā)揮作用，并在 DENV 病毒復(fù)制中發(fā)揮作用。B4GALT7 在我們的屏幕和其他人中被識(shí)別 [ 11 , 12 ]。IL10 亞基 IL10RB 從我們的前 178 個(gè)目標(biāo)列表中類似地確定。IL10 的蛋白質(zhì)水平可能會(huì)影響 COVID-19 疾病的結(jié)果 [ 48 ]。因此，我們測試了這些基因的消耗是否對(duì) SARS-CoV-2 復(fù)制有影響，并且使用 RT-qPCR 分析我們發(fā)現(xiàn)B4GALT7和IL10RB KO 都顯著減少了病毒復(fù)制（補(bǔ)充圖 S1)，表明它們可能在 SARS-CoV-2 感染中起作用。然而，人們可能還記得，使用這種方法，只能確定防止生產(chǎn)性感染的因素。

我們使用我們的全基因組 CRISPR/Cas9 敲除篩選方法準(zhǔn)備了一個(gè)廣泛的數(shù)據(jù)集，從而確定了 SARS-CoV-2 復(fù)制所需的幾種細(xì)胞因子。由于實(shí)驗(yàn)設(shè)計(jì)非常復(fù)雜，因此該研究高度重復(fù)以消除誤報(bào)和隨機(jī)噪聲。我們已經(jīng)使用廣泛的分析和實(shí)驗(yàn)方法確定并驗(yàn)證了選定的因素。所描述的數(shù)據(jù)提供了對(duì) SARS-CoV-2 病毒復(fù)制機(jī)制的有趣見解，并確定了可能作為潛在治療目標(biāo)的因素。

關(guān)鍵詞：

SARS-CoV-2,冠狀病毒,CRISPR-Cas9,細(xì)胞因子,感染機(jī)制,病毒病機(jī),SARS-CoV-2, coronavirus, CRISPR-Cas9, cellular factors, mechanisms of infection, viral pathogenesis,