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

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.

Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae

“…2A). For any induction times less than 10 h, the gene drive activity (18,19) remained below 50% (Fig. 3D).…”

“…However, for isolates subjected to galactose induction (5 h), only the two PCRs corresponding to the gene drive allele (A, B) were present; reactions for the target allele were unable to amplify the expected fragment (C, D). A similar analysis was performed for eight clonal isolates from the NES strains (18,19). However, we sampled two isolates that were sensitive and two samples resistant to the SD-HIS condition; diagnostic PCRs illustrated that the target allele was still present for surviving colonies (Fig.…”

“…There are numerous applications of gene drive biotechnology to control and alter biological populations including global challenges such as eliminating insect-borne diseases [16][17][18] . Recent experimental [19][20][21][22][23][24] and computational studies [25][26][27] highlight the potential of GD systems. However, there remain many unknowns surrounding implementation and management of this new technology (including accidental or malicious release of such a system sans any safeguard or inhibitory mechanism).…”

“…Below, A similar analysis of clonal isolates from the NES-containing strains was performed following drive activation (24 h). Two isolates each from(18) and(19) were chosen that were resistant or sensitive to the SD-HIS condition. (F) The gene drive activities from all measured conditions in (C, D) were plotted.…”

A set of novel CRISPR-based integrative vectors for Saccharomyces cerevisiae

“…The CRISPR-Cas9 system has been extensively used to modify the genome in eukaryotes including yeast 7, 8 . It is composed of a Cas9 nuclease that generates a DNA double strand break (DSB), and a guide RNA (gRNA) that precisely recruits the Cas9 nuclease to a target DNA sequence.…”