Clustering Essential Genes And SCRaMbLEing Of A Yeast Chromosome To Study Genetic Interactions

Genome-scale gene knockout is an important approach to the study of global genetic interactions.At present,the genetic interaction network of Saccharomyces cerevisiae has been revealed relatively comprehensively.The systematic gene-knockout and genome-scale genetic analysis provided an important foundation for the study of genetic interactions.However,large-scale analysis of genetic interactions was time consuming and laborious,and it was still a great challenge to investigate complex interactions involving more genes using traditional gene knock-out methods.Introduction of an inducible SCRaMbLE(Synthetic Chromosome Rearrangement and Modification by Lox Psym-mediated Evolution)system into the synthetic yeast genome make it possible to induce the genome rearrangement,which provides an efficient method to generate random deletion mutants.In this paper,the yeast synthetic chromosomeⅢ(synⅢ)was chosen for SCRaMbLE.All 14 essential genes on wild-type chromosomeⅢ(chrⅢ)were assembled at once by homologous recombination in synⅢ yeast to replace the functions of essential genes on the synthetic chromosome.Based on the clustering of essential genes,essential genes on synⅢ were able to be deleted by SCRaMbLE.In addition,different gene orders were designed to cluster these essential genes,and the influence of gene order on gene expression was studied.This paper confirmed that clustering all the essential genes of chrⅢ in yeast can maintain the normal expression of the genes.However,in individual cases,the gene order affects the transcription level of individual genes.SCRaMbLEing of synⅢ with supplementary of the clustered essential genes to explore synthetic lethal interactions.Investigation of 141 SCRaMbLEd strains revealed varied deletion frequencies of synⅢ chromosomal regions.There were hotspots and cold spots for deletion on synⅢ.Among the no deletion detected regions,synthetic lethal interactions were revealed in the region of synⅢ 82-88 kb and 227-244 kb.It was verified by tetrad spores analysis.This study shows that SCRaMbLE with clustered essential genes enables the deletion of essential genes on synⅢ,which enhances streamlining of synthetic yeast chromosome.Moreover,SCRaMbLE provides a novel strategy to uncover complex genetic interactions.