Robustness of biological systems to genetic insults is widespread. Synergistic sick or lethal interactions are observed for over 75% of non-essential genes in Saccharomyces cerevisiae1,2
. Moreover, interactions involving three or more genes (for example, the seven-gene genetic interaction involving oxysterol binding protein genes3
) are likely to outnumber pairwise genetic interactions1
. Gene families with members related by sequence may frequently harbor a multi-gene interaction, but few of the 80 yeast gene families with six or more genes (Supplementary Table 1 online
have been entirely deleted to identify important underlying shared functions.
Functional redundancy complicates pharmaceutical development. For example, adenosine triphosphate-binding cassette (ABC) transporters overlap in the drugs they export. To isolate the activities of individual transporters, a yeast strain (AD12345678; hereafter termed “AD”) was generated with mutations in nine genes, including seven ABC transporter genes 5
. This strain is hypersensitive to many compounds, facilitating many studies of drug mechanism6
and offers a simplified genetic background for characterizing exogenous transporters7
. However, the two ABC transporter gene clades implicated in drug efflux contain a total of 16 genes8,9
, motivating further deletions within this family.
In yeast, gene deletion is accomplished by replacement with a selectable marker via homologous recombination10
. However, engineering strains bearing many unlinked deletions presents challenges: (1) If deletions are sequentially introduced, the time required scales linearly with the number of target genes. (2) If specific subsets of deletions are lethal in combination, different ‘dead ends’ may be reached depending on the order of deletion, so that achieving the greatest tolerable number of deletions may be impracticable. (3) Finally, there are a limited number of useful selectable markers.
All commonly used methods suffer from the first two problems, but some have addressed the third by removing markers before re-use. Unfortunately, each marker-removal method has additional problems. In the hisG
method, flanking bacterial hisG
sequences recombine to excise the deletion cassette11
, but remnant hisG
sequences accumulating in the genome cause mistargeting of subsequently introduced knock-out fragments12,13
. The Cre/lox
method improves marker excision efficiency and leaves a shorter remnant sequence14,15
. However, Cre recombinase also catalyzes massive genome rearrangements between non-adjacent loxP
sites (E. Boles, personal communication 15,16
. In the ‘delitto perfetto’ method, a short fragment with homology specific to each locus is used for excision of each marker17
. Because yeast transformation is required twice per cycle, this method is time-consuming. Here we describe the ‘Green Monster’ technology which addresses all three problems. Using this technology, we generated broadly drug-sensitive ‘ABC16-monster’ strains lacking 16 ABC transporter genes in clades implicated in multi-drug resistance.