Summary West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen followed by a second screen with a sub-library. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death.

The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide ( Dengue is a mosquito-borne flavivirus infection that has emerged as a serious public health problem worldwide. Four serotypes of dengue virus (DEN-1 to DEN-4) are capable of causing human disease varying in severity from acute selflimiting febrile illness to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The plasma leakage, hemorrhagic manifestations, and shock that characterize DHF/DSS are considered to have an immunological basis, as they are more common during secondary infection with a heterologous dengue virus strain (15,28,33). However, severe clinical manifestations can also occur during primary dengue infection, pointing to a contributory role of viral virulence factors. The WHO estimates that more than 20,000 people worldwide, mainly children, die each year from serious complications of dengue. No specific antiviral therapies are currently available for treating the infection, and efforts to develop a safe prophylactic vaccine have been hindered by the complex role of the immune system in disease pathogenesis (39,52,57). Thus, novel treatment strategies that block viral replication and/or to attenuate the exaggerated cytokine response associated with DHF/DSS complications are urgently needed.Potent and specific gene silencing mediated by RNA interference (RNAi) has generated a great deal of interest in development of RNAi as a therapeutic strategy against viral infections (50,54). Many studies have demonstrated the effectiveness of the RNAi approach to suppress flavivirus infection, including dengue virus replication in experimental cell lines (3,23,26,42,60). In addition, the versatility of RNAi could also be exploited to block important host mediators that contribute to dengue pathogenesis. However, the existence of * Corresponding author. Present address: