A standard knockout procedure alters expression of adjacent loci at the translational level

Abstract

The Saccharomyces cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the ‘head-to-head’ orientation of the deleted and neighboring genes, knockout often led to a shift of the transcription start site of the latter, introducing new uAUG codon(s) into the expanded 5′ untranslated region (5′ UTR). In the ‘tail-to-tail’ arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3′ UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as ∼1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.

Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation

“…In this study, we provide evidence that genome-inserted expression cassettes drive bidirectional transcription and the production of divergent transcripts that can repress neighboring genes in yeast, resulting in potent off-target effects, even in carefully designed single-gene studies. Our data also provide a mechanism for the mysterious NGE, identified in analyses of yeast deletion collection studies, and clarify why some loci are susceptible to off-target effects, while some are immune ( Ben-Shitrit et al, 2012 ; Atias et al, 2016 ; Egorov et al, 2021 ; Makeeva et al, 2019 ). We find that expression cassette insertion at DBP1 reduces the translation of neighboring gene MRP51 through a divergent LUTI driven by the bidirectional activity of cassette promoters ( Neil et al, 2009 ; Xu et al, 2009 ; Churchman and Weissman, 2011 ).…”

“…We conclude that resistance cassette insertion at the DBP1 locus is sufficient to cause widespread off-target phenotypes by mis-regulation of Mrp51. These data raised concerns, given the widespread use of expression cassette insertion in genome editing and were consistent with the phenomena of the NGE ( Ben-Shitrit et al, 2012 ; Atias et al, 2016 ; Egorov et al, 2021 ), identified by analysis of deletion collection mutant phenotypes. While these results were disappointing for our study of the function of Dbp1 helicase, they highlighted this locus as a useful context to dissect the mechanism behind, and ideally to fix, these poorly understood cassette-related side effects.…”

“…While flanking both ends of the cassette with transcription terminator sequences was sufficient to prevent off-target mutant phenotypes at the DBP1/MRP51 locus, we wished to identify the root cause of neighboring gene mis-regulation. Previous work had suggested that the unusually high expression level of the kanMX6 cassette could cause gross chromatin changes that lead to local changes in transcription, and the NGE ( Ben-Shitrit et al, 2012 ; Egorov et al, 2021 ). However, the similar off-target respiratory growth phenotype observed when DBP1 was replaced with either the kanMX6 or TRP1 cassettes, driven by the A. gossypii TEF and S. cerevisiae TRP1 promoters, respectively, led us to disfavor this model, as pTRP1 expression is several-fold lower than pTEF .…”

“…Despite the first reports of NGEs over 10 years ago, the original yeast cassettes remain widely used ( Ben-Shitrit et al, 2012 ). One study that analyzed mutants generated with kanMX modules estimated that replacement at as many as 20% of loci may cause neighboring gene mis-regulation, and ~10% of deletion collection strains have been estimated to demonstrate non-specific phenotypes in genetic interaction data that could be attributed to neighboring gene mis-regulation ( Ben-Shitrit et al, 2012 ; Atias et al, 2016 ; Egorov et al, 2021 ). A recent study focused on protein quantification of each yeast deletion collection strain found that one of the most significant factors determining whether a specific protein’s abundance would change following deletion of a gene encoding another protein was whether the genes encoding the protein pair were genomic neighbors ( Messner et al, 2022 ).…”

“…While selection-mediated editing strategies have been used to advance countless discoveries, their utility relies on the assumption that insertion of a selection cassette at one locus will not disrupt the expression of neighboring genes. However, analyses of mutant phenotypes detected in global studies of the collection of strains in which each non-essential yeast ORF is replaced with a kanMX cassette revealed effects that appeared to result from neighboring gene mis-regulation, termed ‘the neighboring gene effect’ (NGE) ( Ben-Shitrit et al, 2012 ; Atias et al, 2016 ; Egorov et al, 2021 ). This is caused by overlap of the deleted ORF with a regulatory region for the neighboring gene in a subset of cases, as just one of several problems that resulted from the large-scale nature of the effort required to create the deletion collection ( Ben-Shitrit et al, 2012 ; Hughes et al, 2000 ; Teng et al, 2013 ).…”