Identification of critical cellular factors during infection by neurotropic arboviroses through screening with CRISPR-Cas9 libraries

Background: Arboviruses represent a major public health problem. The infection induces an acute and self-limiting febrile condition, however a significant proportion of cases can evolve into serious neurological conditions. The molecular mechanisms involved in infection control versus the evolution of different clinical forms remain unknown Objective: Identify and validate the essential factors involved in infection by the neurotropic arboviruses Chikungunya, Mayaro and Oropouche through large-scale CRISPR screening. Methods and Materials: Here, we used a CRISPR-Cas9 library (Brunello) to identify critical factors and cell damage. The library includes 77,441 guides targeting the deletion of 19,000 human genes, enabling factors involved in the host response to infection. Initially, we generated HuH7 cells that stably express the Cas9 enzyme through transfection. HuH7-Cas9 cells were infected with Mayaro (MAYV), Chikungunya (CHIKV) and Oropouche (OROV) viruses to determine MOI (Multiplicity of Infection) and collection time. HuH7-Cas9 cells were then transduced with lentivirus from the Brunello library. The knockout set of cells was exposed to sublethal infection with MAYV and OROV. Results and Discussion: After scanning and sequencing the infected HuH7-Cas9 cells, 995 positively enriched genes were found in the OROV infection and 987 genes in the MAYV infection. The reads obtained from the sequencing were mapped against the human genome set available in a database. Genes with an absolute value of log2 fold change equal to or >1 and with a significant p-value, correct FDR value were selected for ontology analysis. The initial investigation showed the participation of genes involved in biological and molecular processes, which can be used as possible therapeutic targets.