Genome-wide and Targeted Screening Methods Reveal Host Antiviral Mechanisms to Limit Norovirus Infection

The goal of this thesis was to identify and characterize novel endogenous antiviral genes and to elucidate their mechanisms within the context of murine norovirus (MNoV) infection using cutting-edge techniques. Our studies provide a resource for understanding enteric viral pathogenesis, demonstrate robust methodologies for broadly identifying novel endogenous antiviral molecules, and potentially pave the way for future therapeutics. Here, we describe genome-wide CRISPR activation (CRISPRa) screens performed in both naturally permissive murine BV2 cells and MNoV-permissive engineered HeLa cells which revealed high-scoring gene candidates that inhibit MNoV infection when overexpressed at the endogenous locus. TRIM7, an E3 ubiquitin ligase, emerged as the strongest hit in both screens, emphasizing its potent activity against MNoV regardless of cellular context. We further characterized the molecular mechanisms underlying its antiviral effect and have found TRIM7 to act early in the viral life cycle to limit viral nonstructural protein levels by interacting directly with the viral protease. We also performed an arrayed interferon stimulated gene (ISG) overexpression screen with the goal of uncovering novel anti-MNoV ISGs. This screen was performed in STAT1-deficient MNoV-permissive human fibroblasts. Cells overexpressing a single ISG were challenged with MNoVCW3, and the effect of the ISG on viral infection was quantified by flow cytometry. Our screen identified multiple antiviral ISGs including KCTD14, thought to potentially mediate the activity of the CUL-3 ubiquitin ligase. We found that that KCTD14 inhibits MNoV replication after viral entry, likely by disrupting formation of the replication complex via interaction with viral protein NS3. Data produced via these varied screening methods provide an entirely new lens by which to understand norovirus biology – via both genome-wide and targeted ISG interrogation - leading to the discovery of novel anti-NoV genes that may be leveraged in the future to limit NoV infections

The 15 - 40 Connection meets ASSISTments: Can technology be used to build awareness and change behavior patterns in teens and young adults?

The mission of the 15-40 Connection is to create awareness that there have been close to no improvements in teen and young adult cancer survival rates since 1975, to promote health awareness and to provide the advantage of early detection. ASSISTments is a free web-based tutoring platform by WPI which provides teachers and students with immediate feedback from the use of problem sets containing flexible pre-built and custom content. A team of students is combining these organizations by building cancer problem sets and conducting a study in order to identify successful messages that promote cancer awareness and behavior change. This might just be the best way to educate today's youth - to help them help themselves - and give them a fighting chance against cancer

Creation of Novel Protein Variants with CRISPR/Cas9-Mediated Mutagenesis: Turning a Screening By-Product into a Discovery Tool

<div><p>CRISPR/Cas9 screening has proven to be a versatile tool for genomics research. Based on unexpected results from a genome-wide screen, we developed a CRISPR/Cas9-mediated approach to mutagenesis, exploiting the allelic diversity generated by error-prone non-homologous end-joining (NHEJ) to identify novel gain-of-function and drug resistant alleles of the MAPK signaling pathway genes MEK1 and BRAF. We define the parameters of a scalable technique to easily generate cell populations containing thousands of endogenous allelic variants to map gene functions. Further, these results highlight an unexpected but important phenomenon, that Cas9-induced gain-of-function alleles are an inherent by-product of normal Cas9 loss-of-function screens and should be investigated during analysis of data from large-scale positive selection screens.</p></div

Performance of sgRNAs targeting MAP2K1 in drug resistance screens in A375 cells.

<p>Performance of sgRNAs targeting MAP2K1 in drug resistance screens in A375 cells.</p

Validation of gain-of-function MAP2K1 alleles.

<p>A375 cells constitutively expressing lentiviral-delivered ORFs were assessed for ATP levels after 96 hours of drug treatment, normalized to no drug. Negative controls are shown in gray, positive controls in green, and test ORFs in blue. For vemurafenib, the doses used were, left to right, 0, 0.5, 1, 2, 6, 8, and 10 μM. For selumetinib, the doses used were, left to right, 0, 0.05, 0.1, 0.200, 0.600, 1, and 2 μM.</p

Screening of a pooled sgRNA library tiling across MEK1.

<p>(A) Enrichment of individual sgRNAs targeting MEK1 (MAP2K1) when treated with the BRAF-inhibitor, vemurafenib. Dotted line indicated two standard deviations from the mean of 100 non-targeting sgRNAs. (B) As in panel A but cells were treated with the MEK-inhibitor, selumetinib. (C) Comparison of MEK1 variants enriched by vemurafenib and selumetinib in A375 cells. The average enrichment across three replicates is shown and dotted lines indicate two standard deviations from the mean of 100 non-targeting sgRNA. (D) Comparison of selumetinib-resistant variants of MEK1 between A375 and MEL-JUSO cells. The average enrichment across three replicates is shown and dotted lines indicate two standard deviations from the mean of 100 non-targeting sgRNA.</p

Large variation generated by an individual sgRNA can be used to select for gain-of-function alleles.

<p>(A) Schematic of the experimental approach to examine variants generated by sgRNA targeting and identify gain-of-function alleles. (B) For each of three conditions, variants detected by deep sequencing of the MAP2K1 locus were ranked by their abundance and the reads per million is plotted. Left panel shows all the variants, right panel enlarges one region. (C) Alignment and abundances of selected MAP2K1 alleles generated in untreated or vemurafenib-selected A375 cells by an individual sgRNA targeting MAP2K1 at the site encoding K59. Mismatched and inserted nucleotides are shown in red. Variants that are studied in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170445#pone.0170445.g002" target="_blank">Fig 2</a> are labeled in blue. Variants are ranked by within-sample abundance.</p

Orthologous CRISPR-Cas9 enzymes for Combinatorial Genetic Screens

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current approaches suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from S. aureus and S. pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The “Big Papi” (Paired aureus and pyogenes for intereactions) approach described here will be widely applicable for the study of combinatorial phenotypes