Genome-wide CRISPR-CAS9 knockout library screening for rubella virus-entry factors in JAR4 cells

Human choriocarcinoma JAR4 clone, an MAVS-gene- and PKR-gene-knockout derivative from JAR cells (ATCC HTB-144), was infected with lentiviral pools of human GeCKOv2 CRISPR knockout pooled library (Addgene pooled library #1000000048, #1000000049). One hundred million of JAR4-GeCKOv2-Lib cells were inoculated with the Rubella virus TO-336Vac-717AG1 strain at an MOI of 10. After 3 days of incubation at 35°C, A-1331852 was added to the medium at a concentration of 1 µM to enhance apoptosis induced by rubella virus infection. The surviving cells were allowed to expand, passaged five times and incubated for 1 month. The cells were then inoculated with VSVGFP-RV/CE2E1 at an MOI of 1 twice with a 1-week interval. The genomic DNA was extracted from ten million of the original library cells (control cells) or the selected cells. These screenings were performed in duplicate. Amplicons containing sgRNA integrants from two control cells and two selected cells were prepared by 10 forward primers with different lengths of spacer nucleotides and one each of the four reverse primers with different index sequences using PrimeSTAR GXL DNA Polymerase (Takara Bio, Kusatsu, Japan). 4 nM of Purified PCR products from four different templates was mixed and applied for deep sequencing using MiSeq Reagent Kit v3 (150 cycles) (Illumina, San Diego, CA) in the MiSeq system (Illumina).