Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens

Abstract

Existing antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune response to mycobacterial infection. We used CRISPR/Cas9-mediated high-throughput genetic screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Gué rin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus, high-throughput functional genomic screens can elucidate highly complex hostpathogen interactions and serve to identify HDTs with the potential to improve TB treatment.

High-throughput CRISPR screens to dissect macrophage-Shigellainteractions

“…This result indicated that infection with S. flexneri could be utilized as selective pressure for subsequent host survival-based genetic screens. Independent biological triplicates of genome-wide CRISPR knockout and CRISPRi screen libraries were prepared in THP-1 cells expressing Cas9 and dCas9-Krab, respectively (Doench et al, 2016; Lai et al, 2020; Sanson et al, 2018) ( Figure 1A ). After S. flexneri infection, surviving THP-1 cells with specific single guide RNA (sgRNA) barcodes were maintained in culture medium for continuous replication and harvested for next generation sequencing and analysis.…”

“…We next designed and prepared secondary CRISPR knockout and CRISPRi screen libraries targeting 372 human genes (Lai et al, 2020), in order to validate genome-wide screen hits, to test genes that were associated with different types of host cell death, and to compare the performance between CRISPR knockout and knockdown libraries by ensuring that there were consistent numbers of sgRNAs per gene in each type of library ( Figure 1A ). Similar to our genome-wide screens, surviving THP-1 cells were harvested following S. flexneri infection, and the results of the screens were visualized with volcano plots ( Figures S4A and S4B ).…”

“…The human monocyte cell line THP-1 was a gift from Jianzhu Chen (Singapore-MIT Alliance for Research and Technology). Cas9-expressing and dCas9-Krab-expressing THP-1 cell lines were constructed in previous study (Lai et al, 2020). HEK293FT cells were gifts from Asha Shekaran (Engine Biosciences).…”

“…The Dolcetto human CRISPRi pooled library was a gift from John Doench (the Broad Institute, also available on Addgene #92385). Both secondary CRISPR knockout and CRISPRi libraries, with 10 sgRNAs per gene, were designed to target the 251 genes scored in primary genome-wide screens (133 of these genes were identified as being involved in S. flexneri infection) and 121 genes from literature (47 of these genes were found to be involved in S. flexneri infection) (Lai et al, 2020). 1000 non-targeting sgRNAs were used as controls (Lai et al, 2020).…”

Human stem cell‐based models for studying host‐pathogen interactions

“…Novel insights into previously unrecognised mechanisms of pathogenesis were successfully revealed through the use of CRISPR screening techniques (Chang, Jin, Jiao, & Galan, 2019;Lai et al, 2020;R. J.…”