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

Human choriocarcinoma JAR cells (ATCC HTB-144) were infected with lentiviral pools of human GeCKOv2 CRISPR knockout pooled library (Addgene pooled library #1000000048, #1000000049). One hundred million of JAR-GeCKOv2-Lib cells were inoculated with the Rubella virus Cendhill strain at an MOI of 10. The cells were incubated with medium containing 1 µM ABT-737 at 35°C for approximately 7 days.The surviving cells were passaged, inoculated with Rubella virus again and further incubated for 7 days. 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).

人类绒癌细胞JAR（ATCC HTB-144）经携带人源GeCKOv2 CRISPR敲除混合文库（Addgene混合文库编号#1000000048、#1000000049）的慢病毒池感染。将1亿个JAR-GeCKOv2-Lib细胞以感染复数（MOI）10的剂量接种风疹病毒Cendhill毒株。随后将细胞置于含1 μM ABT-737的培养基中，于35℃条件下孵育约7天。收集存活细胞进行传代，再次接种风疹病毒后继续孵育7天。从1000万份原始文库细胞（对照组细胞）或筛选获得的细胞中提取基因组DNA。该筛选实验设置两次生物学重复。针对2份对照组细胞与2份筛选组细胞的含单引导RNA（sgRNA）整合位点的扩增子，采用10条带有不同间隔核苷酸长度的正向引物，以及4条各携带独特标签序列（index）的反向引物各1份，结合PrimeSTAR GXL DNA聚合酶（宝日医生物Takara Bio，日本滋贺县）进行制备。将来自4种不同模板的纯化PCR产物以4 nM浓度混合后，使用MiSeq系统（因美纳Illumina，美国加利福尼亚州圣地亚哥）搭配MiSeq Reagent Kit v3（150个循环）开展深度测序。