Application of CRISPR libraries for screening cellular factors associated with arbovirus neuroinfection

Abstract

Arboviral diseases represent a serious public health problem that has worsened in recent years due to the introduction of new viruses into highly susceptible populations. The concurrent circulation of different viruses places a heavy burden on healthcare systems, which are ill-prepared to handle the various clinical manifestations of these infections. It is known that arbovirus infections typically result in an acute febrile illness, but a significant portion of infections progresses to severe neurological complications. In this context, the molecular mechanisms that govern infection control versus the development of different clinical forms remain unknown. The present proposal aims to assess the applicability of a CRISPR-Cas9 library as a tool for identifying critical factors for infection and cellular damage caused by various arboviruses of interest. This library consists of 77,441 guides designed for the deletion of approximately 19,000 human genes. This way, we can conduct a global screening of the human genome for factors related to the host response to experimental arbovirus infection through CRISPR/Cas9-mediated gene deletion in permissive cells. After constructing cell clones expressing the Cas9 enzyme constitutively and transducing the library by lentivirus, we will expose the knockout cell populations to lethal infection with Zika, Chikungunya, and Mayaro virus isolates. Finally, we will determine genes and pathways involved in viral replication and cellular damage by sequencing surviving cells and identifying the guides present in that population. The identified genes will be ranked and grouped according to the cellular pathway or process they participate in using gene annotation tools (Gene Ontology). Functional assays for the identified genes will subsequently be conducted, including but not limited to: i) viral load assessment assays for genes encoding receptors and/or surface proteins, ii) cell death assessment assays for genes involved in apoptosis signaling or other forms of cell death, iii) profiling of inflammatory cytokines and chemokines for genes involved in inflammation activation. In this way, the present proposal may contribute to identifying molecular pathways involved in arbovirus infection, potentially yielding new insights into the virus-host interaction processes, which, in turn, could lead to the discovery of potential new therapeutic targets. (AU)