11 Jan 2023
by Richa

Summary- paper 2: Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation

Emily Nicole Powers, Charlene Chan, Ella Doron-Mandel, Lidia Llacsahuanga Allcca, Jenny Kim Kim, Marko Jovanovic, Gloria Ann Brar

eLife, 2022

Questions/gaps addressed:

  • Selection-cassette based genetic engineering (aka Longtine strategy) is a routine strategy in yeast- are there negative consequences associated with this approach?

  • Do selection-mediated editing strategies in yeast lead to transcriptional interference by long undecoded transcript isoforms (LUTIs) of neighboring genes (neighboring gene effect or NGE)? How can this effect be avoided?

Major hypotheses:

  • Insertion of expression cassettes commonly used to edit the genomes of yeast cells, can induce the repression of neighboring genes though synthetic and constitutive LUTI-based repression

Key methods:

  • Ribosome profiling and mRNA-sequencing (mRNA-seq)

  • Label-free mass spec on fractionated polysomes and hierarchical clustering to assess global differences in polysome composition

  • Rapid amplification of cDNA ends (5′RACE)

Major takeaways:

  • Stable cassette-driven divergent transcripts (in most selection marker cassettes TRP1/Kan/Hyg/Nat) were detected in more than 30% of cassette-inserted loci

  • Bidirectional promoter activity from the TEF promoter most likely cause of LUTI-based repression​

  • Bidirectional promoters are very common, including pTEF in yeast, and the CMV, eEF1α, and SV40 promoters in mammalian systems

  • Rrp6/exosome-mediated degradation can mask the divergent transcripts produced from cassette insertion in many cases​

  • Placement of strong transcription terminator sequences flanking both ends of the resistance cassette prevents neighboring gene disruption​, provide modified Longtine based cassettes with these changes.