Global gene disruption in human cells to assign genes to phenotypes by deep sequencing

Abstract

Insertional mutagenesis in a haploid background can lead to complete disruption of gene function1. Here we generate a population of human cells that contain insertions in >98% of their expressed genes. We established Phenotypic Interrogation via Tag Sequencing (PhITSeq) as a method to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of selected cells rather than individual clones provides a rapid assessment of the spectrum of genes involved in phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic gram-negative bacteria that cause tissue damage at distinct anatomical sites2. We identified 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. While related CDTs may share host factors, they also exploit unique host factors yielding a characteristic profile for each CDT.

The adherens junctions control susceptibility to Staphylococcus aureus α-toxin

“…To discover host factors required for α-toxin cytotoxicity, we conducted a live/dead genetic screen by intoxicating haploid human cells (HAP1) carrying knockout alleles in essentially all genes through insertional mutagenesis (22,23). A large-scale library of ∼100 million HAP1 mutagenized cells was treated with recombinant α-toxin, and gene-trap insertion sites were identified in the pool of surviving, toxin-resistant cells.…”

“…HAP1 cells were mutagenized with a retroviral gene trap to cause inactivating mutations throughout the genome, and a haploid genetic screen was performed as previously described (22,23). For a complete description of the haploid genetic screen, see SI Materials and Methods.…”

“…To advance understanding of how S. aureus α-toxin modulates host cell biology, we conducted a high-throughput genetic screen using human cells (22,23) to discover novel host factors required Significance Staphylococcus aureus is a major cause of invasive bacterial infection. One prominent virulence factor is α-toxin, a protein that injures the cell by forming a damaging pore across the cell membrane.…”

Attachment ofChlamydia trachomatisL2 to host cells requires sulfation

“…The cells grow adherently, and are susceptible to infection with and killed by C. trachomatis L2. To find genes important for Chlamydia cytotoxicity, we mutagenized HAP1 cells with a retroviral genetrap vector, yielding a heterogeneous cell population with inactivating insertions in nonessential genes containing on average one insertion event per cell (13,15). Approximately 10 8 mutagenized HAP1 cells were inoculated with C. trachomatis L2 at an multiplicity of infection (MOI) of ∼6 and incubated for 5 d, after which 99.99% of the starting pool of mutagenized cells died.…”

“…We performed a loss-of-function genetic screen in human haploid cells to identify mutant cell lines resistant to Chlamydia (13)(14)(15)(16). This system avoids the potential off-target effects and incomplete knockdowns of an siRNA screen.…”