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Dual transposon sequencing (Dual Tn-seq) to probe genome-wide genetic interactions

Zik, J. J.; Price, M. N.; Arkin, A. P.; Deutschbauer, A. M.; Sham, L. T.

2024-09-25 microbiology
10.1101/2024.09.24.614635 bioRxiv
Show abstract

Understanding gene function on a genome-wide scale is a fundamental goal in biology. With the advent of next-generation sequencing, high-throughput methods such as transposon sequencing (Tn- seq) were developed to measure fitness of single deletion mutants en masse. Nevertheless, gene redundancy complicates these approaches, as inactivating genes individually may not produce discernable phenotypes. Here, we report Dual Tn-seq, a technique for simultaneously assaying fitness of a comprehensive pool of double mutants. Dual Tn-seq couples random barcode transposon-site sequencing (RB Tn-seq) with the Cre-lox system, allowing us to determine the fitness of {approx}68% of all possible double mutant combinations in the human pathogen Streptococcus pneumoniae. Genetic interactions identified from {approx}1.4 billion double mutants uncovered new avenues in widely conserved biochemical pathways, exemplified by the discovery of a new CTP synthase and a regulator of peptidoglycan biosynthesis. Since Dual Tn-seq has very few requirements, it can be readily adapted to diverse organisms.

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