Genome-scale CRISPR knockout screen for Dengue, Japanese Encephalitis, and Human Coronavirus 229E viruses
Genome-scale CRISPR knockout screen for Dengue, Japanese Encephalitis, and Human Coronavirus 229E viruses
For DENV and JEV, a pool of ~40 million mutagenized cells – about 500-fold coverage for each unique sgRNA from the library – were used for each of the screen. The seeded mutagenized cells were subsequently infected with DENV or JEV at a MOI of 0.2 and 0.05 respectively the next day. An additional round of infection was carried out at 18 days post infection. Resistant cells were harvested in two batches for each screen, one at 21 days post infection and the other at 27 days post infection. Cells harvested in the second batch were also reinfected at 21 days post initial infection. A total of 44 million non-infected mutagenized cells were simultaneously harvested to serve as the unselected control. For HCoV-229E screen, a pool of ~60 million mutagenized cells were subsequently seeded and infected with HCoV-229E at a MOI of 0.003. Resistant cells were harvested at 15 days post infection. A total number of 40 million non-infected populations of mutagenized cells were harvested in parallel as the unselected control. Genomic DNA were extracted from each harvested cell pellet using QIAamp DNA Mini Kit (Qiagen). The sgRNA containing sequence were PCR amplified and barcoded with Illumina next generation sequencing indexes.