Genome-wide C-SWAT library for high-throughput yeast genome tagging

Meurer, Matthias; Duan, Yuanqiang; Sass, Ehud; Kats, Ilia; Herbst, Konrad; Buchmuller, Benjamin; Dederer, Verena; Huber, Florian; Kirrmaier, Daniel; Štefl, Martin; Laer, Koen Van; Dick, Tobias P.; Lemberg, Marius K.; Khmelinskii, Anton; Levy, Emmanuel D.; Knop, Michael

doi:10.1038/s41592-018-0045-8

Cited by 68 publications

(108 citation statements)

References 29 publications

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“…In these all genes were modified in the same manner, i.e. by gene deletion or by tagging with a fluorescent protein or affinity tag (Gavin et al, 2002;Ghaemmaghami et al, 2003;Huh et al, 2003;Meurer et al, 2018;Winzeler et al, 1999). We believe that in mammalian cell culture similar endeavors are now at reach using the approach presented in this study.…”

Section: Discussionmentioning

confidence: 99%

“…Obviously, this constitutes a compromise, and includes the possibility that important gene regulatory sequences are omitted from the tagged gene. While for mammalian cells no global data set about the regulatory impact of the 3'-UTR on gene expression is available, data from yeast, where seamless tagging was compared with tagging using a generic 3'-UTR, demonstrated that only about 11% of the genes were impacted in their expression more than 2-fold (Meurer et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

“…We reasoned that the cytoplasmic fluorescence could originate from extra-chromosomal DNA molecules or fragments that have integrated at chromosomal off-target loci. To investigate the fate of the transfected fragments we specifically amplified from cells 3 days after transfection the junctions between tagging cassettes and their upstream flanking DNA sequences using Anchor-Seq (Meurer et al, 2018;Buchmuller et al, 2019) (Fig. 2a).…”

Section: Diffuse Cytoplasmic Fluorescence Is Caused By Unstable Extramentioning

confidence: 99%

“…(a) Anchor-Seq (Meurer et al, 2018) is based on a target enrichment procedure that uses an oligo in the mNeonGreen gene to enrich adjacent sequences for analysis by next generation sequencing using a paired end sequencing protocol (reads 1 & 2).…”

Section: Figure 2 Analysis Of the Fate Of The Transfected Pcr Cassetmentioning

confidence: 99%

“…Sequencing libraries for experiments to determine cassette junction sites presented in Fig. 2 were prepared based on our previously published Anchor-Seq protocol (Meurer et al, 2018) with some modifications to the adapter design to include unique molecular identifiers (UMIs) ( Table S4) (Buchmuller et al, 2019). Quantified libraries were sequenced paired-end with 300 cycles on a NextSeq 550 sequencing system (Illumina) with a spike-in of 20% phiX gDNA library (Illumina).…”

Section: Next-generation Sequencing Of Genomic Dna With Anchor-seqmentioning

confidence: 99%

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CRISPR-Cas12a-assisted PCR tagging of mammalian genes

Fueller

Herbst

Meurer

et al. 2018

Preprint

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AbstractHere we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g. GFP), a Cas12a CRISPR RNA for cleavage of the target locus and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artefacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein tagged genes

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Diffuse Cytoplasmic Fluorescence Is Caused By Unstable Extramentioning

confidence: 99%

Section: Figure 2 Analysis Of the Fate Of The Transfected Pcr Cassetmentioning

confidence: 99%

Section: Next-generation Sequencing Of Genomic Dna With Anchor-seqmentioning

confidence: 99%

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CRISPR-Cas12a-assisted PCR tagging of mammalian genes

Fueller

Herbst

Meurer

et al. 2018

Preprint

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Identification of uncharacterized proteins potentially localized to mitochondria (UPMs) in Saccharomyces cerevisiae using a fluorescent protein unstable in the cytoplasm

Horiuchi

Namba

Saeki

et al. 2021

Yeast

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Eukaryotic cells are composed of organelles, and each organelle contains proteins that play a role in its function. Therefore, the localization of a protein, especially to organelles, is a clue to infer the function of that protein. In this study, we attempted to identify novel mitochondrially localized proteins in the budding yeast Saccharomyces cerevisiae using a fluorescent protein (GFPdeg) that is rapidly degraded in the cytoplasm. Of the budding yeast proteins predicted to localize to mitochondria by the prediction tool Deeploc-1.0, those with known mitochondrial localization or functional relevance were eliminated, and 95 proteins of unknown function were selected as candidates for analysis. By forced expression of GFPdeg fusion proteins with these proteins and observation of their localization, we identified 35 uncharacterized proteins potentially localized to mitochondria (UPMs) including 8 previously identified proteins that localize to mitochondria. Most of these had no N-terminal mitochondrial localization signal and were evolutionarily young "emerging genes" that exist only in S. cerevisiae. Some of these genes were found to be upregulated during the postdiauxic shift phase when mitochondria are being developed, suggesting that they are actually involved in some mitochondrial function.

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High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein Timers

Fung

Blöcher-Juárez

Khmelinskii

2022

Methods in Molecular Biology

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Tandem fluorescent protein timers (tFTs) are versatile reporters of protein dynamics. A tFT consists of two fluorescent proteins with different maturation kinetics and provides a ratiometric readout of protein age, which can be exploited to follow intracellular trafficking, inheritance and turnover of tFT-tagged proteins. Here, we detail a protocol for high-throughput analysis of protein turnover with tFTs in yeast using fluorescence measurements of ordered colony arrays. We describe guidelines on optimization of experimental design with regard to the layout of colony arrays, growth conditions, and instrument choice. Combined with semi-automated genetic crossing using synthetic genetic array (SGA) methodology and high-throughput protein tagging with SWAp-Tag (SWAT) libraries, this approach can be used to compare protein turnover across the proteome and to identify regulators of protein turnover genome-wide.

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Genome-wide C-SWAT library for high-throughput yeast genome tagging

Cited by 68 publications

References 29 publications

CRISPR-Cas12a-assisted PCR tagging of mammalian genes

CRISPR-Cas12a-assisted PCR tagging of mammalian genes

Identification of uncharacterized proteins potentially localized to mitochondria (UPMs) in Saccharomyces cerevisiae using a fluorescent protein unstable in the cytoplasm

High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein Timers

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