Journal: Foods

Article Title: Reduction of Ethyl Carbamate in an Alcoholic Beverage by CRISPR/Cas9-Based Genome Editing of the Wild Yeast

doi: 10.3390/foods12010102

Figure Lengend Snippet: Strains and plasmids used in this study.

Article Snippet: pCas9-NAT , Cas9 expression plasmid, Nourseothricin marker , Addgene (#43802).

Techniques: Isolation, RNA Expression, Plasmid Preparation, Marker, Expressing

Journal: Biology Methods & Protocols

Article Title: An enChIP system for the analysis of genome functions in budding yeast

doi: 10.1093/biomethods/bpac025

Figure Lengend Snippet: Expression of 3xFLAG-Sp-dCas9 from the 3xFLAG-dCas9/pTEF1p-CYC1t plasmid in budding yeast. ( A ) Schematic illustration of the 3xFLAG-dCas9/pTEF1p-CYC1t plasmid expressing 3xFLAG-Sp-dCas9. The TEF1 constitutive promoter drives transcription of 3xFLAG-dCas9 fused with the SV40 nuclear localization signal (NLS) and the CYC1 terminator. ( B ) Expression of 3xFLAG-Sp-dCas9, detected via Simple Western™ assay with an anti-FLAG Ab, in 10 randomly selected YPH499 clones transformed with the 3xFLAG-dCas9/pTEF1p-CYC1t plasmid. CBB staining is shown as a protein loading control. (-): the parental YPH499 strain. ( C ) Quantification of expression levels of 3xFLAG-Sp-dCas9 in the selected clones. Background noise was subtracted from each signal. Standard deviation is shown. ( D ) Expression of 3xFLAG-Sp-dCas9 in a representative clone (#1). Expression of 3xFLAG-Sp-dCas9 was detected via immunoblotting with an anti-FLAG Ab (left panel). CBB staining is shown as a protein loading control (right panel). An extra band corresponding to 3xFLAG-Sp-dCas9 is visible in the 3xFLAG-dCas9/pTEF1p-CYC1t-transformed clone (#1) (arrowhead) (-): the parental YPH499 strain.

Article Snippet: To construct a plasmid expressing 3xFLAG-Sp-dCas9 in budding yeast (3xFLAG-dCas9/pTEF1p-CYC1t: Addgene, Watertown, MA, USA, Cat# 62190; RRID: Addgene_62190), the p414-TEF1p-Cas9-CYC1t plasmid (a gift from the George Church laboratory; Addgene, Cat# 43802; RRID: Addgene_43802) [ ] was digested with SpeI (Takara Bio, Kusatsu, Japan, Cat# 1086A) and MluI (Takara Bio, Cat# 1071A), and treated with bacterial alkaline phosphatase ( Escherichia coli C75) (Takara Bio, Cat# 2120A).

Techniques: Expressing, Plasmid Preparation, Western Blot, Clone Assay, Transformation Assay, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Episomal editing of synthetic constructs in yeast using CRISPR

doi: 10.1101/2022.06.21.496881

Figure Lengend Snippet: a . The two-plasmid system. Cas9 plasmid is pre-transformed into yeast, followed by a second transformation of the sgRNA plasmid. b . The single-plasmid system. Cas9 and sgRNA are expressed from a single plasmid, with CEN/ARS (up) or 2μ backbone (bottom).

Article Snippet: The original Cas9 and gRNA expression modules were modified from p414-TEF1p-Cas9-CYC1t (Addgene# 43802) and p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene# 43803).

Techniques: Plasmid Preparation, Transformation Assay

Journal: bioRxiv

Article Title: Episomal editing of synthetic constructs in yeast using CRISPR

doi: 10.1101/2022.06.21.496881

Figure Lengend Snippet: a . Editing efficiency targeting genomic ADE2 , as determined by three replicates. Error bars represent mean ± SD. ns, not significant as analyzed with unpaired t test. b . Survival rate of colonies transformed with Cas9/sgRNA alone without donor DNA. Error bars represent mean ± SD, n=3 and p<0.0001 (****), as analyzed with unpaired t test. c . Spot assay on SC–Ura plates with BY4741 strains carrying Cas9 expressed from CEN/ARS or 2μ plasmids. The pRS416 ( CEN-URA3 ) and pRS426 ( 2μ-URA3 ) are used as empty plasmid controls. d . Doubling time of the same yeast strains used in the spot assay. Error bars represent mean ± SD, n=3 and p<0.001 (***), as analyzed with unpaired. Growth curves are shown in Supplementary Figure 1. e . Method to test the Cas9 toxicity and plasmid stability. f . Proportion of colonies lost with URA3 plasmid. Error bars represent mean ± SD, n=3 and p<0001 (****), as analyzed with unpaired t test.

Article Snippet: The original Cas9 and gRNA expression modules were modified from p414-TEF1p-Cas9-CYC1t (Addgene# 43802) and p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene# 43803).

Techniques: Transformation Assay, Spot Test, Plasmid Preparation

Journal: bioRxiv

Article Title: Episomal editing of synthetic constructs in yeast using CRISPR

doi: 10.1101/2022.06.21.496881

Figure Lengend Snippet: a . Structure of episomal mSox2 assembly. b . Episomal editing strategy. Cas9 creates a DSB at CTCF8 which can be repaired with a donor DNA containing CTCF8Δ. c . Editing efficiency with one-plasmid (CEN or 2μ backbone) or two-plasmid system (CEN/2μ) as determined by genotyping using deletion-specific primers (Supplementary Figure 2). Error bars represent mean ± SD. n=3 and p<0.05 (*), as analyzed with unpaired t test. d . Survival rate of colonies transformed with Cas9/gRNA target CTCF8 without donor DNA templates. Error bars represent mean ± SD. n=3 and p<0.005 (**), as analyzed with unpaired t test. WGS read coverage of colonies with unintended or successful edits aligned to the mSox2 assembly. f . Unintended deletion boundaries from mapped to the indicated mouse genome repetitive elements (see also Supplementary Table 1).

Article Snippet: The original Cas9 and gRNA expression modules were modified from p414-TEF1p-Cas9-CYC1t (Addgene# 43802) and p426-SNR52p-gRNA.CAN1.Y-SUP4t (Addgene# 43803).

Techniques: Plasmid Preparation, Transformation Assay