Abstract
Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome. The development of molecular toolkits and “integration cassettes” have provided fungal systems with a collection of strategies for tagging, deleting, or over-expressing target genes; typically, these consist of a C-terminal tag (epitope or fluorescent protein), a universal terminator sequence, and a selectable marker cassette to allow for convenient screening. However, there are logistical and technical obstacles to using these traditional genetic modules for complex strain construction (manipulation of many genomic targets in a single cell) or for the generation of entire genome-wide libraries. The recent introduction of the CRISPR/Cas gene editing technology has provided a powerful methodology for multiplexed editing in many biological systems including yeast. We have developed four distinct uses of the CRISPR biotechnology to generate yeast strains that utilizes the conversion of existing, commonly-used yeast libraries or strains. We present Cas9-based, marker-less methodologies for (i) N-terminal tagging, (ii) C-terminally tagging yeast genes with 18 unique fusions, (iii) conversion of fluorescently-tagged strains into newly engineered (or codon optimized) variants, and finally, (iv) use of a Cas9 “gene drive” system to rapidly achieve a homozygous state for a hypomorphic query allele in a diploid strain. These CRISPR-based methods demonstrate use of targeting universal sequences previously introduced into a genome.
Highlights
Part of the success of this model organism includes the development and utility of genome-wide libraries—collections of separate yeast strains each containing a unique modification—that can be used to screen all non-essential genes that would be required for different molecular processes
Given the recent expansion and utility of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing across many model systems, we sought to employ this technology to provide the yeast community with a new multipurpose molecular toolkit for strain construction
Previous work has provided a suite of useful gene-tagging cassettes for S. cerevisiae with the majority focused on either (i) biochemical epitope tags (Tagwerker et al, 2006; Moqtaderi and Struhl, 2008; Funakoshi and Hochstrasser, 2009) or (ii) fluorescent protein variants (Sheff and Thorn, 2004; Lee et al, 2013; Malcova et al, 2016)
Summary
Part of the success of this model organism includes the development and utility of genome-wide libraries—collections of separate yeast strains each containing a unique modification (an engineered plasmid, an integrated epitope tag, or deletion of a gene, etc.)—that can be used to screen all non-essential (or essential) genes that would be required for different molecular processes. The set of available yeast libraries has expanded to include epitope tags (Ross-Macdonald et al, 1999; Ghaemmaghami et al, 2003), over-expression arrays (Sopko et al, 2006; Ho et al, 2009), fluorescent protein fusions (Huh et al, 2003), gene deletions (Giaever et al, 2002), and essential hypomorphic alleles (Breslow et al, 2008; Li et al, 2011) These collections have been useful in uncovering cellular, biochemical, and genetic interactions across numerous fields. Construction of such a large collection (∼5,000 separate strains) still presents many logistical challenges
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