Global gene disruption in human cells to assign genes to phenotypes

Insertional mutagenesis in a haploid background can lead to complete disruption of gene function(1). 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 sites(2). 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.