Use of a ubiquitous gene-editing tool in budding yeast causes off-target repression of neighboring gene protein synthesis

Precision genome-editing approaches have long been available in budding yeast, enabling introduction of gene deletions, epitope tag fusions, and promoter swaps through a selection-based strategy. Such approaches allow loci to be modified without disruption of coding or regulatory sequences of neighboring genes. Use of this approach to delete DBP1 however, led to silencing of expression and the resultant loss of function for the neighboring gene MRP51. We found that insertion of a resistance cassette to delete DBP1, drove a 5’ extended alternative transcript for MRP51 which dampened Mrp51 protein synthesis. Misregulation of MRP51 occurred through an integrated transcriptional and translational repressive long undecoded transcript isoform (LUTI)-based mechanism that was recently shown to naturally regulate gene expression in yeast and other organisms. Cassette-induced MRP51 repression drove all mutant phenotypes we detected in cells deleted for DBP1. Selection cassette-mediated aberrant transcription events are not specific to this locus or a unique cassette but can be prevented by insertion of transcription insulators flanking the cassette. Our study suggests the existence of confounding off-target mutant phenotypes resulting from misregulated neighboring loci following genome edits in yeast. Furthermore, features of LUTI-based regulation are broadly conserved to eukaryotic organisms which indicates the potential that similar misregulation could be unnoticed in other edited organisms as well.