Influenza A is a single-stranded, multi-segmented, negative sense RNA virus of the family Orthomyxoviridae and is the causative agent of seasonal Influenza. Influenza viruses cause significant impacts on a global scale regarding public health and economics. Annual influenza virus infections in the United States account for over 200,000 hospitalizations, up to 49,000 deaths, and an $87.1 billion economic burden. Influenza A virus has caused several pandemics since the turn of the 20th century. The effects of Influenza on public health and economics, compounded with low efficacy of the annual vaccine and emerging antiviral resistance, brings to light the need for an effort to stem these impacts, prevent pandemics, and protect public health by developing novel treatments. This project proposes an alternative approach to combatting Influenza by targeting host factors hijacked during infection that, if inhibited, significantly impair viral RNA expression, but result in low host toxicity. The host factors we examined include RNA export factors (XpoT and Xpo5) and RNA helicases (UAP56 and URH49). We selected paralogs URH49 (DDX39A) and UAP56 (DDX39B) because previous studies suggest differing roles during infection, but we theorize that their high degree of sequence similarity, similar function, and association with many of the same cellular factors may allow them to substitute for one another if one is inhibited. CRISPR was considered as the primary method to evaluate the effect of knockout of these factors on viral RNA expression and host cell toxicity. CRISPR is an RNA-guided mechanism for gene editing and can be used to make null mutations in targeted host genes. However, CRISPR proved to be a significant challenge and, while we could not conclusively confirm whether the CRISPR plasmids were effective at targeting our genes of interest, our initial results were not promising and we did not pursue this approach further. As an alternative, host RNA export factors were evaluated using siRNA to knockdown the factor prior to influenza infection. RNA was analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The potential of inhibiting UAP56 or URH49 as a novel therapeutic target was determined using a visual assessment of cell death. We found that siRNA-mediated knockdown of XpoT and Xpo5 did not have any impact on viral RNA synthesis early during infection. siRNA against UAP56 and DDX39 (targets both UAP56 and URH49) resulted in significant impairment in viral RNA synthesis, confirming previously established work suggesting that UAP56 and URH49 have important roles during infection. Importantly, these helicases play an interferon (IFN) independent role to enhance viral replication, as indicated by analysis in IFN deficient VERO cells. A viability assay relying on trypan blue exclusion did not yield trustworthy results, so a visual assessment of cell death was done. The visual assessment confirms previously-established observations that Nxf1 siRNA treatments result in a high degree of cell death, indicating the toxic nature of Nxf1 inhibition. Cells treated with UAP56 or DDX39 siRNAs demonstrated little to no additional toxicity compared to the non-target control, suggesting they can be inhibited to serve as antiviral targets.

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Nucleases are an essential component of the prokaryotic CRISPR-Cas immunity as well as repair mechanisms within prokaryotic organisms. To better understand the adaptation step of CRISPR-Cas immunity, I have characterized three Cas4 proteins from hyperthermophilic archaea: SSO0001 and SSO1391 from Sulfolobus solfataricus and Pcal_0546 from Pyrobaculum calidifontis. All three proteins have metal-dependent 5′ to 3′ exonuclease and endonuclease activities, while SSO1391 also demonstrates 3′ to 5′ exonuclease activity. Site-directed mutagenesis confirmed that the conserved RecB motif residues are important for the nuclease activity in all three proteins. SSO0001 and Pcal_0546 also exhibit ATP-independent unwinding and cleavage of splayed arm substrates. Structural analysis of SSO0001 showed it is a toroidal decamer with a [4Fe-4S] cluster and Mn2+ ion bound in the active site located inside the internal tunnel. Our results show that Cas4 proteins have the ability to create 3'-DNA overhangs which may contribute to the addition of novel CRISPR spacers.

Nucleases are an essential component of the prokaryotic CRISPR-Cas immunity as well as repair mechanisms within prokaryotic organisms. To better understand the adaptation step of CRISPR-Cas immunity, I have characterized three Cas4 proteins from hyperthermophilic archaea: SSO0001 and SSO1391 from Sulfolobus solfataricus and Pcal_0546 from Pyrobaculum calidifontis. All three proteins have metal-dependent 5′ to 3′ exonuclease and endonuclease activities, while SSO1391 also demonstrates 3′ to 5′ exonuclease activity. Site-directed mutagenesis confirmed that the conserved RecB motif residues are important for the nuclease activity in all three proteins. SSO0001 and Pcal_0546 also exhibit ATP-independent unwinding and cleavage of splayed arm substrates. Structural analysis of SSO0001 showed it is a toroidal decamer with a [4Fe-4S] cluster and Mn2+ ion bound in the active site located inside the internal tunnel. Our results show that Cas4 proteins have the ability to create 3'-DNA overhangs which may contribute to the addition of novel CRISPR spacers.

Powerful mechanisms of genetic interference in both unicellular and multicellular organisms are based on the sequence-directed targeting of DNA or RNA by small effector RNAs. In many bacteria and almost all archaea, RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are involved in an adaptable and heritable gene-silencing pathway. Resistance to phage infection is conferred by the incorporation of short invading DNA sequences into the prokaryotic genome as CRISPR spacer elements separated by short repeat sequences. A central aspect to this pathway is the processing of a long primary transcript (pre-crRNA) containing these repeats by crRNA endonucleases to generate the mature effector RNAs that interfere with phage or plasmid gene expression. Here we describe a structural and functional analysis of the CasE endonuclease of T. thermophilus a member of the Ecoli CRISPR sub-type. High resolution X-ray structures of CasE bound to repeat RNAs model both the pre-and post-cleavage complexes associated with processing the pre-crRNA. These structures establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene-silencing.

Cachexia is a multifactorial metabolic wasting syndrome that affects a large percentage of cancer patients and results in the involuntary loss of skeletal muscle and adipose tissue. The consequences of this condition include metabolic imbalances and fatigue, which are strongly associated with poor prognosis. While the specific mechanism for skeletal muscle wasting is still undefined, LIF secreted by C26 colon carcinoma cells has recently be found to induce atrophy in treated myotubes. The purpose of this study is to determine the necessity of LIF for inducing atrophy in mouse myotubes by producing a knockout of Lif in C26 cells using CRIPSR-Cas9. Media was collected from these cells and used to treat myotubes. Measurements of myotube diameters were made and atrophy was compared between myotubes that received medium from C26 and C26Lif-/- cells. A dosage of recombinant mouse LIF was also added to LIF-deficient medium in order to determine if LIF alone was sufficient to induce atrophy. At study endpoint, myotubes that were treated with media taken from C26 cells showed significant signs of atrophy compared to myotubes that were treated C26Lif-/- media. LIF was also shown to be sufficient to induce myotube atrophy on its own, with atrophy being rescued in myotubes that received a dosage of LIF added to C26Lif-/- media. These results demonstrate that LIF is required for atrophy to be induced in mouse myotubes treated with media taken from cancer cells, and can do so independent of other secreted factors.

Viruses are ubiquitous in natural growth environments and cause severe losses to crop yields, globally. Approximately 30% of plant viruses described to date are grouped within the family Potyviridae, making it one of the largest plant virus families. Furthermore, certain potyvirus species can cause devastating diseases in several agriculturally and economically important crops. Hence, gaining insight into potyvirus resistance and recovery mechanisms in plants is an important research focus. This thesis firstly explores how environmental cues can modulate the activity of a central form of viral defence, namely RNA silencing. Specifically, high temperatures and low light intensities were found to increase the efficacy of viral RNA silencing in Arabidopsis, resulting in recovery from infection by Turnip Mosaic Virus. The biological context and potential for agricultural exploitation of these phenomena are discussed. Secondly, this thesis explores the ability to engineer resistance alleles using the latest genome editing techniques. Specifically, resistance to Turnip Mosaic Virus was successfully engineered in Arabidopsis by CRISPR/Cas9-induced deletion of a known susceptibility factor eIF(iso)4E. Biotechnological methods to implement this proof of concept research in crop species were also investigated.

Several techniques have recently been described for precise mutagenesis of selected target sites in the genome. This thesis establishes the method of gene targeting by CRISPR/Cas system in D. melanogaster and compares it with gene targeting using TALENs. To test the mutagenesis systems, we choose an endogenous gene encoding concentrative nucleoside transporter gene (CNT1). We have received two mutants containing large deletions affecting the N-terminal part of the CNT1 gene. We show that CRISPR/Cas is useful tool for targeted gene disruption in D. melanogaster.

8q24.21 is a highly amplified region in cancer and associated with many epithelial cancer such as bladder, breast, colorectal and prostate cancer. The proto-oncogene c-myc is located in this region and surrounded by many lncRNAs genes such as PCAT family, CCAT family, PRNCR1. In this study, we used CRISPR-Cas9 constructs to knock out PCAT1, PRNCR1, CASC8, CASC11 and also the sequences between PCAT1-CASC11 and CASC8-CASC11in the prostate cancer cell PC3. The transfected cells with CRISPR-Cas9 targeting CASC11 gene had less proliferation ability comparing with the transfected cells with CRISPR-Cas9 targeting PCAT1, PRNCR1 or CASC8. The role of CASC11 in cancer progression and development is obscure. In our study, The CASC11 Knockout efficiency was 90% compare to the control cell. Furthermore, the study showed the importance of CASC11 in cell proliferation by significantly decreasing in the forming colonies and the growth rate comparing to the control. Also, MMP2, MMP3 and MMP9 expression levels were detected in the transfected cell by using real time PCR and the result revealed the crucial role for CASC11 in metastasis and migration. The slug and vimentin expression levels were reduced in the transfected and the double transfected clones which indicate the possible role of CASC11 in epithelial mesenchymal transition and cell motility. Taken together, our study revealed that the lncRNA CASC11 plays important roles in prostate cancer progression and metastasis by promoting the cell proliferation and migration.

Loss-of-function genetic screens are a powerful approach to identify the genes involved in biological processes. For nearly a century, forward genetic screens in model organisms have provided enormous insight into many cellular processes. However, the difficulty in generating and recovering bi-allelic mutations in diploid cells severely hindered the performance of forward genetic screens in mammalian cells. The development of a retroviral gene-trap vector to mutagenise the human near-haploid KBM7 cell line transformed forward genetic screens in human cells. The re-purposing of the microbial CRISPR/Cas9 system now offers an effective method to generate gene knockouts in diploid cells. Here, I performed a head-to-head comparison of retroviral gene-trap mutagenesis screens and genome-wide CRISPR knockout screens in KBM7 cells. The two screening approaches were equally effective at identifying genes required for the endoplasmic reticulum (ER)-associated degradation of MHC class I molecules. The ER-resident enzyme HMG-CoA reductase (HMGCR) catalyses the rate-limiting step in the cholesterol biosynthesis pathway and is targeted therapeutically by statins. To maintain cholesterol homeostasis, the expression of HMGCR is tightly regulated by sterols transcriptionally and post-translationally. Sterols induce the association of HMGCR with Insig proteins, which recruit E3 ubiquitin ligase complexes to mediate degradation of HMGCR by the ubiquitin proteasome system. However, the identity of the E3 ligase(s) responsible for HMGCR ubiquitination is controversial. Here, I use a series of genome-wide CRISPR knockout screens using a fluorescently-tagged HMGCR exogenous reporter and an endogenous HMGCR knock-in as an unbiased approach to identify the E3 ligases and any additional components required for HMGCR degradation. The CRISPR screens identified a role for the poorly characterised ERAD E3 ligase RNF145. I found RNF145 to be functionally redundant with gp78, an E3 ligase previously implicated in HMGCR degradation, and the loss of both E3 ligases was required to significantly inhibit the sterol-induced degradation and ubiquitination of HMGCR. A focused E3 ligase CRISPR screen revealed that the combined loss of gp78, RNF145 and Hrd1 was required to completely block the sterol-induced degradation of HMGCR. I present a model to account for this apparent complexity.