Genome engineering to create targeted gene deletions, mutations, reporter constructs, and epitope-tagged proteins is a key strategy for the mechanistic dissection of almost any biological process. Budding yeast was the first eukaryotic organism in which this became highly facile, thanks to the development of a one-step PCR-based editing strategy, frequently used with a shared toolkit of selection cassettes (Longtine et al., 1998; Wach et al., 1997; Wach et al., 1994; Baudin et al., 1993; Lorenz et al., 1995). These tools became common, routinely used by thousands of labs as well as to enable large global endeavors, such as creation of the yeast deletion collection (Giaever et al., 2002). Because of the high fidelity, ease of use, and rapid nature of this selection-mediated strategy, it has remained commonplace despite the development of methods allowing non-selection-marked mutations that include CRISPR/Cas9-based editing (Jinek et al., 2012; Güldener et al., 1996; Gray et al., 2005).

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). However, most cases of the NGE remain unexplained, and this lack of mechanistic understanding makes it unclear how prevalent this effect is in traditional small-scale laboratory studies, and how to prevent it.

It is now understood that genomic loci can have complex and linked transcriptional outputs, even in yeast. For example, activity from one transcription start site (TSS) can interfere with the output of nearby TSSs in an adjacent sense or antisense configuration, and that most, if not all, promoters are bidirectionally active (Teodorovic et al., 2007; Neil et al., 2009; Preker et al., 2008; Core et al., 2008; Xu et al., 2009; Seila et al., 2008; Churchman and Weissman, 2011; Jin et al., 2017). Transcription interference is seen in diverse organisms, including yeast and human cells, and can occur between TSSs driving coding or non-coding RNAs and controlling both transcript isoform identity and transcript levels (Chia et al., 2017; Hirschman et al., 1988; Martens et al., 2004; van Werven et al., 2012; Hongay et al., 2006; Hausler and Somerville, 1979; Adhya and Gottesman, 1982; Proudfoot, 1986; Boussadia et al., 1997; Emerman and Temin, 1984; Corbin and Maniatis, 1989; Struhl, 1985). It has also been shown that more than one TSS is often present at a single locus, even in the simple budding yeast (Pelechano et al., 2013).

Transcription interference between TSSs within the same genomic locus is an important feature of a naturally occurring type of regulation dependent on long undecoded transcript isoforms (LUTIs; Chia et al., 2017; Chen et al., 2017). For genes regulated by this strategy, 5′ extended LUTIs are transcribed in place of canonical mRNAs to temporally downregulate protein synthesis (Chia et al., 2017; Chen et al., 2017; Cheng et al., 2018; Van Dalfsen et al., 2018). Here, use of an upstream alternate TSS represses transcription from the canonical TSS in cis via transcriptional interference (Chia et al., 2017). In concert, translation of the main ORF-encoded protein products from LUTIs are repressed by translation of competitive AUG-initiated upstream ORFs (uORFs) (Chen et al., 2017; Wethmar, 2014; Barbosa et al., 2013; Hinnebusch et al., 2016; Law et al., 2005; Brar et al., 2012; Cheng et al., 2018). Natural LUTI-based regulation has been shown to modulate gene expression for many genes during meiosis and the unfolded protein response in yeast (Chen et al., 2017; Cheng et al., 2018; Van Dalfsen et al., 2018). Furthermore, features of LUTI-based regulation are highly conserved across eukaryotes, and evidence suggests the presence of this regulation in more complex species, such as humans and algae (Hollerer et al., 2019; Sehgal et al., 2008; Moseley et al., 2002). Here, we show that insertion of expression cassettes commonly used edit the genomes of yeast cells, can induce the repression of neighboring genes though synthetic and constitutive LUTI-based repression.

In particular, we report that selection-cassette replacement of the ORF for DBP1 causes mis-regulation of the adjacent gene, MRP51, despite no changes to the MRP51 coding or regulatory regions, as a result of synthetic LUTI-based repression. All phenotypes we observed in cells with a cassette-mediated deletion of the DBP1 ORF were caused by mis-regulation of MRP51, rather than loss of DBP1, consistent with an NGE. We leveraged the strong off-target effects observed with cassette-mediated replacement of DBP1 to interrogate the mechanism driving this phenomenon. We find that cassette-mediated off-target effects were general to all expression cassettes that we inserted at the DBP1 locus and are driven by the bidirectional cassette promoter activity now understood to be inherent to eukaryotic promoters.

These data point to an undesirable feature of expression cassette insertion that is currently being overlooked in the design of genome-editing approaches. While we find that stable cassette-driven divergent transcripts were detected in ~30% of cassette-inserted loci, all cassette-inserted loci have detectable divergent transcripts in cells lacking nuclear exosome-mediated RNA decay. Thus, our data suggest that divergent transcription results ubiquitously from expression cassette insertion, but features—including transcription termination sequences adjacent to cassette insertion—can naturally mitigate off-target neighboring gene mis-regulation. Because it is difficult to predict from sequence information alone whether a locus will be sensitive to neighboring gene mis-regulation, we designed improved expression cassettes containing an additional terminator sequence flanking the promoter, which prevents neighboring gene disruption. Use of such cassettes should improve the specificity of engineered mutant strains for future studies. More broadly, our study uncovers a mechanism by which genome engineering can drive neighboring gene mis-expression, and that warrants consideration in the design of future studies in yeast, as well as other eukaryotes.

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). Production of this divergent transcript drives both transcription interference and uORF-based translation repression for MRP51 (Chia et al., 2017; Chen et al., 2017; Cheng et al., 2018). Because most, if not all, eukaryotic promoters are thought to be bidirectional, these data suggest that any inserted expression cassette containing a promoter could drive neighboring off-target effects (Teodorovic et al., 2007; Neil et al., 2009; Preker et al., 2008; Core et al., 2008; Xu et al., 2009; Seila et al., 2008).

How commonly does expression cassette insertion cause confounding mutant phenotypes? Confidence in the specificity of yeast genome-editing cassettes has motivated their widespread use, and many landmark studies defining the functions of conserved proteins have been performed using this approach. It is routine for multiple loci to be disrupted within the same strain to enable analysis of genetic interactions, and global yeast deletion, hypomorphic, and epitope tag collections were created by use of the kanMX insertion cassette (Giaever et al., 2002; Winzeler et al., 1999; Giaever and Nislow, 2014; Chu and Davis, 2008; Ghaemmaghami et al., 2003; Huh et al., 2003; Breslow et al., 2008). 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). It is important to note that all previously reported examples of the NGE, including those noted here, focused only on integrations of kanMX modules (Ben-Shitrit et al., 2012; Atias et al., 2016; Egorov et al., 2021; Makeeva et al., 2019; Messner et al., 2022). Our study determined that NGEs are driven by cassettes of disparate sequence. This observation allowed us to determine that the promoter bidirectionality that is inherent to eukaryotic promoters, rather than any feature of a particular cassette sequence, drives neighboring gene disruption. Thus, any expression cassette can cause disruptive NGEs, suggesting a much larger pool of potentially affected mutants than previously considered.

In fact, we found cassette-driven divergent transcripts to be produced at 100% of expression cassette-inserted loci analyzed, but they were often short and masked by exosome-mediated RNA decay. This suggests that additional features local to cassette insertion can prevent off-target phenotypes. Given that bidirectional promoter activity originating from cassettes drives neighboring gene disruption, there are two non-mutually exclusive explanations for why some loci would produce disruptive divergent transcripts and others would not. First, the different chromatin context at inserted loci may affect the bidirectional promoter activity (Churchman and Weissman, 2011; Jin et al., 2017; Struhl, 1985; Porrua and Libri, 2015; Marquardt et al., 2014). Alternatively, bidirectional activity and divergent transcript production could occur at all loci, with off-target effects being prevented in many cases by local sequence characteristics. Our data do not exclude the possibility that chromatin context is a factor, but do suggest that the role of local sequence features in determining the span of divergent transcription is important. One such feature that may naturally prevent neighboring gene disruption at cassette-inserted loci are NNS termination signals, like those known to terminate and prevent endogenously produced divergent transcripts from interfering with neighboring genes (Steinmetz et al., 2001; Schulz et al., 2013; Porrua and Libri, 2015). Both NNS termination signals and canonical coding gene transcription termination sequences were sufficient to prevent to ‘insulate’ bidirectional promoters within selection cassettes inserted at the DBP1 locus.

How do divergent transcripts from cassette-inserted loci drive off-target phenotypes? While we focused on the integrated transcription and translation regulation driven by LUTI-based disruption of MRP51 in this study, mis-regulation of adjacent genes by cassette-driven transcription interference alone may be even more frequent. LUTI-based neighboring gene disruption requires the adjacent gene to be in a ‘head-to-head’ orientation, the presence of repressive uORFs upstream of the coding sequence, and a continuous divergent transcript containing the entire ORF of the repressed gene (Chia et al., 2017; Chen et al., 2017; Cheng et al., 2018). Cassette-driven transcription interference, however, can occur regardless of the presence of repressive uORFs upstream of the neighboring coding sequence, does not require the divergent transcript to be continuous over the neighboring ORF, and can act on genes in either orientation to the inserted cassette (Chia et al., 2017; Hirschman et al., 1988; Martens et al., 2004; van Werven et al., 2012; Hongay et al., 2006; Hausler and Somerville, 1979; Adhya and Gottesman, 1982; Proudfoot, 1986; Boussadia et al., 1997; Emerman and Temin, 1984; Corbin and Maniatis, 1989). For example, in haploid cells, induction into meiosis is blocked by the expression of two separate non-coding RNAs that repress an antisense mRNA (RME2/IME4) or a downstream sense RNA (IRT1/IME1) via transcription interference (Hongay et al., 2006; van Werven et al., 2012). Transcription interference between loci can be effective over large distances, like the ~1800 nt span over which transcription of the repressive IRT1 long non-coding RNA represses IME1 (van Werven et al., 2012).

The type of cassette-mediated off-target effect in this study is difficult to detect or predict by sequence alone. No unexpected mutations exist in these cases and even mRNA measurements of neighboring genes are not diagnostic unless all possible transcript isoforms are measured from a given locus. Additionally, our data suggest that at as many as 70% of cassette-inserted loci, divergent transcripts are produced but degraded by the nuclear exosome, meaning that even the lack of an observable stable divergent transcript is not sufficient to conclude wild-type regulation from a cassette-adjacent gene because disruption can occur regardless of the transcript’s stability. Measuring translation or protein levels for adjacent genes should be diagnostic of this undesirable side effect, but ribosome profiling is not a routine experiment that is applied to most mutants, and tagging proteins can alter function and regulation. Additionally, as many genes are expressed in a condition-specific manner, measurement of the adjacent gene’s protein levels compared to wild-type may give false confidence unless the proper conditions are chosen. Consistently, we observed condition-specific manifestation of the cassette-driven divergent transcription at the DBP1/MRP51 locus. For 3/5 additional potential NGE cases with detectable aberrant transcripts in exosome proficient cells (Figure 4), adjacent genes were lowly expressed, even in wild-type cells under the conditions surveyed. Considering the number of factors affecting whether cassette adjacent genes are mis-regulated, we argue that it is currently not possible to predict when and where cassette insertion would result in mis-regulation severe enough to mislead studies.

Despite this complexity, complementation-based rescue experiments—namely single-copy insertion of the deleted gene with its entire endogenous regulatory region at another locus—allowed us to identify the unexpected regulation in the case of DBP1 disruption, and should be more commonly used to confirm phenotypes. However, for genome-wide experiments, those using cassettes for epitope tagging, or experiments in which genetic interactions between several genes are being investigated, this may not be feasible. Therefore, we created ‘insulated’ resistance cassettes containing transcription terminator sequences flanking the promoter, which prevent off-target effects and possible misinterpretation of mutant phenotypes. Other possible alternatives to avoiding these confounding off-target effects include methods that allow mutants to be isolated without expression cassette insertion. Seamless deletions such as those created with the CRISPR/Cas9 approach avoid cassette insertions, but can still result in changes to the local environment of adjacent genes, potentially resulting in off-target effects (Jinek et al., 2012; Güldener et al., 1996; Gray et al., 2005). For the case of a markerless CRISPR/Cas9-based deletion of the DBP1 ORF, we observed translation of a previously untranslated ORF within the mutant transcript containing only the DBP1 5′ and 3′ UTR (Figure 5—figure supplement 2). One could imagine that in cases like these, translation of novel ORFs could also cause misleading neomorphic mutant phenotypes, not linked to loss of function of the deleted ORF. These data highlight the point that even well controlled genomic changes can have unintended consequences on the translation of neighboring coding sequences, and emphasize the value of careful analysis of gene expression from loci neighboring those that are manipulated.

We note that two-step strategies to isolate mutants free of selection cassettes have been described but are often used the context of allowing subsequent re-use of auxotrophic markers and cassettes (Güldener et al., 1996; Gray et al., 2005). One example of this uses the Cre/loxP system to excise resistance cassettes following mutation isolation (Güldener et al., 1996). While this strategy can prevent neighboring gene disruption by cassettes, it is slower and more labor intensive than a single step process, and thus may not be commonly utilized to its full potential. For example, in two datasets we analyzed for cassette-driven divergent transcripts in strain backgrounds deficient for exosome-mediated RNA decay, cassette integrated mutants contained flanking loxP sites that were not utilized to excise the cassette (Schmidt et al., 2012). Use of a single-step ‘insulated’ cassette for genome editing maintains the efficiency of the originally designed system, while preventing the potentially catastrophic off-target effects that disruption of neighboring gene expression can cause.

While our study was limited to yeast, all the key factors behind the cassette-induced off-target effects described here are conserved in higher eukaryotes. Because of this, we hypothesize that genome-editing-driven transcriptional interference, including LUTI-based repression, is likely to occur in other organisms. It is now understood that promoters in yeast, mammals, and other eukaryotes are inherently bidirectional, but that evolution of local sequences to can limit transcription interference of adjacent genes by transcription termination of divergent transcripts within a short distance of their initiation (Teodorovic et al., 2007; Neil et al., 2009; Seila et al., 2008; Churchman and Weissman, 2011; Jin et al., 2017; Schulz et al., 2013; Arnone, 2020; Villa et al., 2020; Ha et al., 2022; Ntini et al., 2013; Almada et al., 2013; Core et al., 2008; Xu et al., 2009). Although NNS-based termination does not prevent production of long stable divergent transcripts from bidirectional promoters in mammals, analogous evolution of early polyadenylation sites appears to have achieved the same result (Schulz et al., 2013; Ntini et al., 2013; Almada et al., 2013; Porrua and Libri, 2015). In both cases, the local context of promoters defines the degree to which they produce divergent transcripts with the ability to interfere with neighboring gene expression.

The bidirectional nature of commonly used expression cassette promoters, including pTEF in yeast, and the CMV, eEF1α, and SV40 promoters in mammalian systems, has long been known (Curtin et al., 2008; Gidoni et al., 1985). However, consequences of this for their modular use in expression constructs do not yet seem to be widely considered. Early studies that established ‘minimal’ promoters for modular use in expression cassettes explicitly served to separate the transcript-generating feature of promoter regions from their native sequence context. Because local sequence features outside of these minimal promoter regions can limit the effects of divergent transcription, and insertion of a terminator flanking inserted promoter sequences can serve a similar function, our study suggests this simple fix to prevent cassette-based off-target effects in future studies. Our study also emphasizes the need for proper controls to establish causality of observed phenotypes to the intended mutation, even when using precision genome engineering approaches in yeast and other organisms.

mRNA sequencing and ribosome profiling data are available at NCBI GEO, with accession numbers GSE207267 and GSE207189. Mass spectrometry data are available at MassIVE, with accession number MSV000089724.

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Author details

1. Emily Nicole Powers

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4497-7318

2. Charlene Chan

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Ella Doron-Mandel

   Department of Biological Sciences, Columbia University, New York, United States

   Contribution

   Data curation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Lidia Llacsahuanga Allcca

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Jenny Kim Kim

   Department of Biological Sciences, Columbia University, New York, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Marko Jovanovic

   Department of Biological Sciences, Columbia University, New York, United States

   Contribution

   Data curation, Methodology, Project administration

   Competing interests

   No competing interests declared

7. Gloria Ann Brar

   1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
   2. California Institute for Quantitative Biosciences (QB3), University of California, Berkley, Berkley, United States
   3. Center for Computational Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   gabrar@berkeley.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8560-9581

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