Methods

The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

Plasmids and Yeast strains

All plasmids used were from the MyLO Toolkit collection constructed here. They are listed in Table Sl, and will be made available from Addgene. Plasmids were made using Golden Gate cloning or homologous recombination in yeast as outlined in detail in Supplementary Information 1 with primers listed in Table S2. Yeast strains, listed in Table S3, were made with CRISPR/Cas9-based transformations using MyLO Toolkit components. All plasmids were confirmed by sequencing and all integrants were confirmed by PCR.

Fluorescence-based Yeast Transformation Assay

Assays of transformation efficiency were performed by integrating a strong GFP expression cassette (pBBK93, TDH3p Neon(gfp)) into wild type yeast using a lithium acetate-based transformation protocol (Gietz and Schiestl, 2007). Briefly, 3 OD600 log phase yeast were transformed by incubating for 30 min at 30°C and then 42°C in a 150 μL final volume with concentrations as in Gietz et al. Twenty percent of the reaction was plated and incubated two days at 30°C prior to imaging on a Safe Imager 2.0 (Invitrogen) blue light source to identify positive, green colonies. In some cases, colonies were counted using the Fiji (Schindelin et al., 2012) Color Threshold tool followed by the Analyze Particles tool.

Fluorescence Microscopy

Log phase yeast grown in synthetic selective media were imaged on slides using a DMi6000B microscope (Leica Microsystems) with an HCX PL APO 63x oil objective, an Orca R2 CCD camera (Hamamatsu) and Volocity software (PerkinElmer). Images within each panel were evenly exposed and processed using Fiji (Schindelin et aL, 2012) and Photoshop CC (Adobe) except for those in Figure 5 where large fluctuations in intensity necessitated different exposure times.

Western Blot

Log phase yeast were lysed by freezing a pellet, suspending them in Alternate Thorner Buffer (8M Urea, 5% SDS, 40 mM Tris 6.8, 0.1 mM EDTA, 0.4 mg/mL bromophenol blue, 1% β-mercaptoethanol) with glass beads, heating at 70°C for 5 min and then vortexing 1-2 min. An amount of lysate equivalent to 0.1 OD600 of yeast was run on an SDS-PAGE gel, transferred to nitrocellulose paper and blotted with mouse anti-HA (Abeam ab18181) followed by donkey anti-mouse conjugated to IR-Dye 800CW (Mandel Scientific 926-32212). The blot was imaged on an Odessey 9120 Infrared Imager (LI-COR).

Flow Cytometry

Log phase yeast grown in synthetic complete media were measured on an Accuri C6 flow cytometer (BD Biosciences). For each sample 10,000 events were collected and the mean green fluorescence values of the complete ungated populations were recorded.

Article TitleThe MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

Abstract

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 Toolkit with three components: (i) a set of optimized S. pyogenes Cas9-guide RNA (gRNA) expression vectors with five selectable markers and the option to either pre-clone or co-transform the gRNAs; (ii) vectors for the one-step construction of integration cassettes expressing an untagged or GFP/RFP/HA-tagged gene of interest at one of three levels, supporting localization and overexpression studies; and (iii) integration cassettes containing moderately expressed GFP- or RFP-tagged compartmental markers for colocalization experiments. These components allow rapid, high efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle to simplify yeast research.

Competing Interest Statement

The authors have declared no competing interest.


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