Arrayed mutant haploid embryonic stem cell libraries facilitate phenotype-driven genetic screens

Forward genetic screens using mammalian embryonic stem (ES) cells have identified genes required for numerous cellular processes. However, loss-of-function screens are more difficult to conduct in diploid cells because, in most cases, both alleles of a gene must be mutated to exhibit a phenotype. Recently, mammalian haploid ES cell lines were successfully established and applied to several recessive genetic screens. However, all these screens were performed in mixed pools of mutant cells and were mainly based on positive selection. In general, negative screening is not easy to apply to these mixed pools, although quantitative deep sequencing of mutagen insertions can help to identify some ‘missing’ mutants. Moreover, the interplay between different mutant cells in the mixed pools would interfere with the readout of the screens. Here, we developed a method for rapidly generating arrayed haploid mutant libraries in which the proportion of homozygous mutant clones can reach 85%. After screening thousands of individual mutant clones, we identified a number of novel factors required for the onset of differentiation in ES cells. A negative screen was also conducted to discover mutations conferring cells with increased sensitivity to DNA double-strand breaks induced by the drug doxorubicin. Both of these screens illustrate the value of this system.