CRISPR/Cas9-mediated gene knockout screens and target identification via whole-genome sequencing uncover host genes required for picornavirus infection

Kim, Heon Seok,Lee, Kyungjin,Bae, Sangsu,Park, Jeongbin,Lee, Chong-Kyo,Kim, Meehyein,Kim, Eunji,Kim, Minju,Kim, Seokjoong,Kim, Chonsaeng,Kim, Jin-Soo American Society for Biochemistry and Molecular Bi 2017 The Journal of biological chemistry Vol.292 No.25

<P>Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes (<I>i.e.</I> three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery.</P>

Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

Kim, Heon Seok,Lee, Kyungjin,Kim, Seong-Jun,Cho, Sungchan,Shin, Hye Jin,Kim, Chonsaeng,Kim, Jin-Soo Cold Spring Harbor Laboratory Press 2018 Genome research Vol.28 No.6

<P>Pooled CRISPR screens based on lentiviral systems have been widely applied to identify the effect of gene knockout on cellular phenotype. Although many screens were successful, they also have the limitation that genes conferring mild phenotypes or those essential for growth can be overlooked, as every genetic perturbation is incorporated in the same population. Arrayed screens, on the other hand, incorporate a single genetic perturbation in each well and could overcome these limitations. However, arrayed screens based on siRNA-mediated knockdown were recently criticized for low reproducibility caused by incomplete inhibition of gene expression. To overcome these limitations, we developed a novel arrayed CRISPR screen based on a plasmid library expressing a single guide RNA (sgRNA) and disrupted 1514 genes, encoding kinases, proteins related to endocytosis, and Golgi-localized proteins, individually using 4542 sgRNAs (three sgRNAs per gene). This screen revealed host factors required for infection by coxsackievirus B3 (CVB3) from Picornaviridae, which includes human pathogens causing diverse diseases. Many host factors that had been overlooked in a conventional pooled screen were identified for CVB3 infection, including entry-related factors, translational initiation factors, and several replication factors with different functions, demonstrating the advantage of the arrayed screen. This screen was quite reliable and reproducible, as most genes identified in the primary screen were confirmed in secondary screens. Moreover, ACBD3, whose phenotype was not affected by siRNA-mediated knockdown, was reliably identified. We propose that arrayed CRISPR screens based on sgRNA plasmid libraries are powerful tools for arrayed genetic screening and applicable to larger-scale screens.</P>

Ginsenoside Rg3 Restores Hepatitis C Virus-Induced Aberrant Mitochondrial Dynamics and Inhibits Virus Propagation

( Jae Young Jang ),( Seong-jun Kim ),( Eun-jung Kim ),( Eun Kyung Cho ),( Dae-gyun Ahn ),( Chonsaeng Kim ),( Han Seul Park ),( Soung Won Jeong ),( Sae Hwan Lee ),( Sang Gyune Kim ),( Young Seok Kim ) 대한간학회 2017 춘·추계 학술대회 (KASL) Vol.2017 No.1

Aims: Hepatitis C virus (HCV) alters mitochondrial dynamics associated with persistent viral infection and suppression of innate immunity. Mitochondrial dysfunction is also a pathologic feature of direct-acting antiviral (DAA) treatment. Despite the high efficacy of DAAs, their treatment of patients with chronic hepatitis C in interferon-sparing regimens occasionally produces undesirable side effects such as fatigue, migraine and other conditions, which may be linked to mitochondrial dysfunction. Methods: Here we show that clinically prescribed DAAs, including Sofosbuvir, affect mitochondrial dynamics. To counter these adverse effects, we examined HCV- and DAA-induced aberrant mitochondrial dynamics modulated by ginsenoside, which is known to support healthy mitochondrial physiology and the innate immune system. We screened several ginsenoside compounds showing antiviral activity using a robust HCV cell culture system. We investigated the role of ginsenosides in antiviral efficacy, alteration of the mitochondrial transmembrane potential, abnormal mitochondrial fission, its upstream signaling, and mitophagic process caused by HCV infection or DAA treatment. Results: Only one of the compounds, ginsenoside Rg3 (G-Rg3), exhibited the notable and promising anti-HCV potential. Treatment of HCV-infected cells with G-Rg3 increased HCV core protein-mediated reduction in the expression level of cytosolic p21 required for increasing the cyclin-dependent kinase 1 (CDK1) activity, which catalyzes Ser616 phosphorylation of dynamin-related protein 1 (Drp1). The HCV-induced mitophagy, which follows mitochondrial fission, was also rescued by G-Rg3 treatment (figure). Conclusions: G-Rg3 inhibits HCV propagation. Its antiviral mechanism involves restoring the HCV-induced Drp1-mediated aberrant mitochondrial fission process, thereby resulting in suppression of persistent HCV infection.

A Novel Series of Highly Potent Small Molecule Inhibitors of Rhinovirus Replication

Kim, Jinwoo,Jung, Yu Kyoung,Kim, Chonsaeng,Shin, Jin Soo,Scheers, Els,Lee, Joo-Youn,Han, Soo Bong,Lee, Chong-Kyo,Neyts, Johan,Ha, Jae-Du,Jung, Young-Sik American Chemical Society 2017 Journal of medicinal chemistry Vol.60 No.13

<P>Human rhinoviruses (hRVs) are the main causative pathogen for common colds and are associated with the exacerbation of asthma. The wide variety in hRV serotypes has complicated the development of rhinovirus replication inhibitors. In the current investigation, we developed a novel series of benzothiophene derivatives and their analogues (6-8) that potently inhibit the replication of both hRV-A and hRV-B strains. Compound 6g inhibited the replication of hRV-B14, A21, and A71, with respective EC50 values of 0.083, 0.078, and 0.015 mu M. The results of a time-of-addition study against hRV-B14 and hRV-A16 and resistant mutation analysis on hRV-B14 implied that 6g acts at the early stage of the viral replication process, interacting with viral capsid protein. A molecular docking study suggested that 6g has a capsid-binding mode similar to that of pleconaril. Finally, derivatives of 6 also displayed significant inhibition against poliovirus 3 (PV3) replication, implying their potential inhibitory activities against other enterovirus species.</P>

Repurposing Screens of FDA-Approved Drugs Identify 29 Inhibitors of SARS-CoV-2

( Keun Bon Ku ),( Hye Jin Shin ),( Hae Soo Kim ),( Bum-tae Kim ),( Seong-jun Kim ),( Chonsaeng Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2020 Journal of microbiology and biotechnology Vol.30 No.12

COVID-19, caused by the novel coronavirus SARS-CoV-2, has spread globally and caused serious social and economic problems. The WHO has declared this outbreak a pandemic. Currently, there are no approved vaccines or antiviral drugs that prevent SARS-CoV-2 infection. Drugs already approved for clinical use would be ideal candidates for rapid development as COVID-19 treatments. In this work, we screened 1,473 FDA-approved drugs to identify inhibitors of SARS-CoV-2 infection using cell-based assays. The antiviral activity of each compound was measured based on the immunofluorescent staining of infected cells using anti-dsRNA antibody. Twenty-nine drugs among those tested showed antiviral activity against SARS-CoV-2. We report this new list of inhibitors to quickly provide basic information for consideration in developing potential therapies.

Enterovirus inhibitory activity of C-8-<i>tert</i>-butyl substituted 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-<i>e</i>][1,2,4]triazolo[4,3-<i>a</i>]pyrimidin-5(4<i>H</i>)-ones

Kumar Biswas, Bishyajit,Malpani, Yashwardhan R.,Ha, Neul,Kwon, Do-Hyun,Soo Shin, Jin,Kim, Hae-Soo,Kim, Chonsaeng,Bong Han, Soo,Lee, Chong-Kyo,Jung, Young-Sik Elsevier 2017 Bioorganic & medicinal chemistry letters Vol.27 No.15

<P><B>Abstract</B></P> <P>Members of a series of 4-aryl-6,7,8,9-tetrahydrobenzo[4,5]thieno[3,2-<I>e</I>][1,2,4]triazolo[4,3-<I>a</I>]pyrimidin-5(4<I>H</I>)-ones (<B>1</B>, Fig. 2) were prepared and tested against representative enteroviruses including Human Coxsackievirus B1 (Cox B1), Human Coxsackievirus B3 (Cox B3), human Poliovirus 3 (PV3), human Rhinovirus 14 (HRV14), human Rhinovirus 21 (HRV 21) and human Rhinovirus 71 (HRV 71). The C-8-<I>tert</I>-butyl group on the tetrahydrobenzene ring in these substances was found to be crucial for their enterovirus activity. One member of this group, <B>1e</B>, showed single digit micromolar activities (1.6–8.85μM) against a spectrum of viruses screened, and the highest selectivity index (SI) values for Cox B1 (>11.2), for Cox B3 (>11.5), and for PV3 (>51.2), respectively<B>.</B> In contrast, <B>1p</B>, was the most active analog against the selected HRVs (1.8–2.6μM), and showed the highest selectivity indices among the group of compounds tested. The SI values for <B>1p</B> were 11.5 for HRV14, 8.4 for HRV21, and 12.1 for HRV71, respectively.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>