A genome-wide hepatocyte CRISPR-CAS9 screen to identify host factors essential for optimal Plasmodium liver stage development.
Description
To identify host genes critical for Plasmodium LS development, we used the GeCKOv2 sgRNA pool to generate a whole-genome knockout library in HepG2-CD81 cells (Sanjana et al., 2014; Shalem et al., 2014). HepG2-CD81 cells were transduced with lentivirus containing the pooled GeCKOv2 sgRNA library of 114,123 sgRNAs targeting 19,031 protein-coding genes (~6 sgRNAs/gene) and selected in puromycin for 5-7 days. To evaluate sgRNA diversity in the HepG2-CD81-GeCKOv2 library, we PCR-amplified the integrated sgRNA cassettes from genomic DNA extracted from transduced cells and subjected the amplified library to Illumina sequencing. At the gene level, sgRNAs targeting all but 15 of 19,031 (99.92%) protein-coding genes were observed. Twelve to fourteen days after post-transduction, we infected forty million puromycin-resistant cells with a green fluorescent protein (GFP) expressing-Plasmodium yoelii at a multiplicity of infection (MOI) of 0.3. After 24 h of infection, cells were sorted into infected and bystander cell populations by GFP signal intensity with fluorescence-activated cell sorting (FACS). Separately, a parallel culture of uninfected cells was also maintained to normalize the sgRNA frequency distributions. We obtained four independent biological replicates with library generation and sequencing occurring in parallel. Genes with significantly enriched sgRNAs were identified for both the bystander and infected populations when compared to uninfected cells. Cells that harbor genetic alterations restricting P. yoelii development (i.e., sgRNAs that target host genes important for infection) were expected to be enriched in the uninfected bin; we termed this group ‘putative positive regulators of infection’. We categorized sgRNAs enriched in the infected cells as ‘putative negative regulators of infection’. In this initial screen, we identified 242 sgRNAs that were statistically enriched in infected or bystander groups after accounting for multiple hypotheses. There were 67 sgRNAs significantly enriched in the infected cells compared to uninfected cells and 175 genes were significantly enriched in the bystander bin relative to the uninfected bin.