Scarless Gene Tagging with One-Step Transformation and Two-Step Selection in Saccharomyces cerevisiae and Schizosaccharomyces pombe

Abstract

Gene tagging with fluorescent proteins is commonly applied to investigate the localization and dynamics of proteins in their cellular environment. Ideally, a fluorescent tag is genetically inserted at the endogenous locus at the N- or C- terminus of the gene of interest without disrupting regulatory sequences including the 5’ and 3’ untranslated region (UTR) and without introducing any extraneous unwanted “scar” sequences, which may create unpredictable transcriptional or translational effects. We present a reliable, low-cost, and highly efficient method for the construction of such scarless C-terminal and N-terminal fusions with fluorescent proteins in yeast. The method relies on sequential positive and negative selection and uses an integration cassette with long flanking regions, which is assembled by two-step PCR, to increase the homologous recombination frequency. The method also enables scarless tagging of essential genes with no need for a complementing plasmid. To further ease high-throughput strain construction, we have computationally automated design of the primers, applied the primer design code to all open reading frames (ORFs) of the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the fission yeast Schizosaccharomyces pombe (S. pombe), and provide here the computed sequences. To illustrate the scarless N- and C-terminal gene tagging methods in S. cerevisiae, we tagged various genes including the E3 ubiquitin ligase RSP5, the proteasome subunit PRE1, and the eleven Rab GTPases with yeast codon-optimized mNeonGreen or mCherry; several of these represent essential genes. We also implemented the scarless C-terminal gene tagging method in the distantly related organism S. pombe using kanMX6 and HSV1tk as positive and negative selection markers, respectively, as well as ura4. The scarless gene tagging methods presented here are widely applicable to visualize and investigate the functional roles of proteins in living cells.

A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance

“…The basic yTRAP plasmid pGAN147 was constructed by PCR and subsequent Gibson assembly of components into pDML112 (Landgraf et al, 2016) (Figure S1A). Gateway cloning was used to insert genes-of-interest into pGAN147 to form yTRAP fusions.…”

“…Endogenous tagging of yeast genes with mNeonGreen was achieved by homologous recombination with long homology regions (~300 bp) generated by PCR, as previously described (Landgraf et al, 2016). mNeonGreen was amplified with a NAT resistance cassette and assembly PCR was used to add the relevant homology regions at the terminus before transformation.…”

CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains

“…The ability to have a “marker-less” genetic alteration is often desirable, but can also present technical challenges. Prior to the introduction of CRISPR/Cas9, there have been other systems used to remove a selectable marker (often in at least two or more consecutive steps) using integration vectors (Sikorski and Hieter, 1989 ), the Cre-Lox system (Germino et al, 2006 ), or other methods, such as the classic “loop in-loop out” (Landgraf et al, 2016 ; Wang et al, 2017 ). This is often extremely useful in strains where the presence of multiple plasmids and multiple loci have all been (each) marked with the entire ensemble of selectable markers in yeast (or if a particular genetic background does not include the full suite of auxotrophic knockouts).…”

Transient Hsp90 suppression promotes a heritable change in protein translation

“…Vma1-mCherry tagging - The tagging was performed as previously described [54] using pFA6a_mCherry_HPHMX as template and the following primers :…”