Fig. 1

Schematic workflow diagram of CRISPR/Cas9 screen. (1) In the first step, the guide RNAs are either designed in silico or pre-made gRNA libraries are used; then cloning and validation of the gRNA library begins. (2) Packaging the gRNAs into lentiviruses and transducing the target cells with these lentiviruses, which disrupts or leads to gene expression. (3) The pooled mutagenized cell population is then infected with the virus. (4) After the infection, the selection is performed based on the survival or death of the host cells. If the infection occurs with a cytolytic virus, it allows the selection of virus-resistant cells (positive selection) in cell viability-based screens. In these survivors, the knockout of host factors contributing to viral pathogenesis will enable them to survive. Negative screens aim to identify cells that cannot survive the selection pressure. Therefore, it is often necessary to use negative screens to recognize essential genes which their loss of function will not contribute to the cell's survival. Alternatively, fluorescence-activated cell sorting (FACS) can be utilized to investigate persistent or non-cytolytic viruses. (5) Genomic DNA extraction of the selected cells and control cells is conducted, and then PCR amplification occurs. (6) Genes that are enriched or depleted in comparison to the control population are determined using Next-Generation Sequencing (NGS) analysis