Genome-wide CRISPR screening identifies LRP1 as an entry factor for SFTSV
Overall, the Mouse-GeCKOv2-Library-B (Addgene# 1000000053) encompassing 62,804 sgRNAs targeting 20,611 genes generated by the Zhang laboratory was used in this study. To perform the screen, MEF cells stably expressing Cas9 were infected with the sgRNA lentivirus library at an MOI of 0.3 and a library coverage of 600x. 48 h after infection, cells were re-seeded and selected with 2 ug/ml puromycin for 48h. Re-seed the cells and allow them to recover in 6% FBS DMEM without puromycin for 2 days. Then, 2.2 x 108 untreated library cells were collected as the control sample. Another 2.2 x 108 library cells were subjected to SFTSV at MOI=1 as screen sample. A plate of MEF-Cas9 cells without sgRNA library was used as infection control. Five days post infection, when the MEF-Cas9 cells all fell off due to severe CPE, reseed the remaining library cells in screen sample and allow them to recover for 3 days. Re-challenge the re-seeded library cells with SFTSV at MOI=1 and collect the remaining cells 10 days post infection. Genomic DNA was extracted from replicate samples and sgRNA inserts were amplified by PCR. The products of the PCR reactions were subjected to gel electrophoresis on a 1.5% agarose gel and the 247 bp DNA bands were extracted and sequenced at the Novaseq6000 platform (Illumina).