Express dCas9-VP64
Integrate pTPGI_dCas9_VP64

We received plasmid pTPGI_dCas9_VP64 from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas". After glycerol stocks and miniprep, we performed a restriction analysis to check the identity of our plasmid. We linearised the plasmid, in order to integrate it into yeast genome. Only our fourth trial to integrate the plasmid was successful.

Materials and methods

• Glycerol stocks
• Miniprep
• Restriction analysis
• Polymerase Chain Reaction
• Yeast integration
• For more details about our procedures, see here.

Results

We used four different sets of enzymes for the restriction analysis. Linearized pTPGI_dCas9_VP64 is expected to be 10'987 bp. We observe that the gel (fig.1) corresponds to the expected one.
The plasmid was linearised both with EagI HF and NotI HF prior to integration. We integrated each linearised plasmid to obtain two different yeast strains.

Express dCas9-VP64
Western Blot of dCas9-VP64

The Western Blot allows to check the expression of dCas9.

Materials and methods

• Western Blot A AJOUTER SUR PROTOCOLS

Results

Results of Western Blot.

Integrate reporter plasmid
Linearise reporter plasmid

We received the plasmid pCYC1m_yeGFP from Addgene. The plasmid was found from the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas".

Materials and methods

• PCR (cf. Protocols). After glycerol stocks and minipreps. we linearised the plasmid by PCR according to the following procedure.

Results

Integrate reporter plasmid
Synthesize promoters

From the article "Tunable and multifunctional eukaryotic transcription factors based on Crispr/Cas", we learnt that different regions on the promoters could lead to activation or inhibition when dCas9-VP64 was bound. We chose to modify the region of strongest activation, named c3, and the two regions that lead to the strongest inhibition, c6 and c7. We synthesized promoters CYC_0, CYC_1, CYC_2 and CYC_3. They only differ between one another by the three regions c3, c6 and c7. The promoter CYC_0 is the original promoter, already present in the plasmid pCYC1m_yeGFP.

Results

Four different promoters according to Fig. 2.

Integrate reporter plasmid
Add Gibson overlaps by PCR

We amplified the promoters CYC_0, 1, 2, 3 by PCR adding the Gibson overlaps in order to assemble each fragment in the linearised plasmid pCYC1m_yeGFP.

Materials and methods

• PCR (cf. Protocols). The PCR was performed according to the following procedure. The PCR for CYC_3 was repeated a second time, and a third time, due to a frameshift detected by sequencing.

Results

On the gel electrophoresis that we ran after PCR, we observe the three CYC fragments at the right height (Fig.3). These fragments are then used for the Gibson assembly in the plasmid pCYC_yeGFP.

Integrate reporter plasmid
Gibson assembly of CYC promoters

The promoters CYC_0, 1, 2, 3 have overlaps from previous PCR. They were assembled in the linearised plasmid pCYC1m_yeGFP.

Materials and methods

• Gibson assembly was performed according to the following procedure. The Gibson assembly for CYC_3 was repeated a second time, and a third time
• Colony PCR was performed according to the following procedure. The colony PCR was repeated a second time for CYC1, a third, fourth, fifth and sixth time for CYC3.

Results

The results of the Gibson assembly are given by a colony PCR. The amplicon should be 308 bp since we used primers f_Gbs_CYC and r_Gbs_CYC. We observe on Fig. 4 that CYC2 on lane 5 and CYC3 on lanes 8 and 9 migrated at the right height. These colonies can be sent to sequencing. Colony PCR was repeated to find CYC1.

After colony PCR, sequencing indicated a frameshift in the CYC3 construct. This is why we repeated the whole process, from the start, for CYC3.

Integrate reporter plasmid
Integrate the reporter plasmids

The four constructs, pCYC_0_yeGFP, pCYC_1_yeGFP, pCYC_2_yeGFP, pCYC_3_yeGFP, are ready to be integrated in yeast genome.

FUN FACT: on September 8, ten days before wiki freeze, we changed our strain of yeast. The strain we used previously, 4271???, had a mutation on GAL4 gene, preventing its growth in galactose supplemented medium and preventing induction of GAL1 promoter.The new strain is W303.

We present our methods, both for the former strain and the more recent one.

Materials and methods

• Integration (cf. Protocols)
• Plasmids were linearized with restriction enzyme EagI-HF.
• After integration, yeasts were plated on the following selection plates: CYC0, CYC1 and CYC2.

• Polymerase Chain Reaction (cf. Protocols). After glycerol stocks and minipreps, the PCR with primers f_Gbs_ADH1 and r_Gbs_ADH1 was performed according to the following procedure.

Results

The size of p405ADH1 plasmid is 7.2 kbp. Altough it is not in frame with the ladder, a different migration rate is observed between the PCR products (p405ADH1 linearized) and the circular plasmid on the 10th lane. Therefore, we assumed that the PCR was successful.

Integrate and express gRNAs
Synthesize gRNA-expressing cassette

The design of the gRNA-expressing cassettes is inspired by the article "Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells". The activating gRNA, c3, and the two repressing gRNAs, c6 and c7, were synthesized. Each gRNA is fused to the HH ribozyme on the 5' end, and to the HDV ribozyme on the 3' end.

Results

According to Fig.8, we synthesized the activating gRNA cassette c3 and the two repressing gRNA cassettes c6 and c7. Each of these gRNAs: c3, c6, c7, is in four exemplaries. The numbering 0, 1, 2, 3, corresponds to promoter CYC_0, CYC_1, CYC_2, CYC_3.

Integrate and express gRNAs
PCR amplify the gRNA-expressing cassettes

The gRNA-expressing cassettes c3_0, c3_1, c3_2, c3_3 (activating sequences), c6_0, c6_1, c6_2, c6_3, (inhibiting sequences), c7_0, c7_1, c7_2, c7_3 (inhibiting sequences). The c3 gRNA expressing cassettes were synthesized along with their corresponding CYC promoter as individual G-Blocks, all other gRNAs were synthesized as individual G-Blocks. Four primers were used in order to PCR out or amplify the gRNAs and the promoters: f_IDT_tri, f_IDT_squ, r_IDT_dia and r_IDT_cir. For detailed reaction mixes, click here LIEN A INSERER

Materials and methods

• Polymerase Chain Reaction

Results

Each amplicon (gRNA expressing cassette or CYC promoter) is 250 bp long (the exact length can vary by max two bp depending on the primers used). Several PCRs were required for amplification, we only show the gels corresponding to the PCR products we kept.
Fig...: c6_0, c6_1, c6_2, c6_3, CYC_0, CYC_1, CYC_2
Fig...: c7_0, c7_1, c7_2
Fig...: c7_3
Fig...: CYC_3, c3_1, c3_2, c3_3
Fig...: c3_0

Integrate and express gRNAs
PCR out DsRed2

In order to verify the expression of the gRNAs cassettes, we used fluorescent protein DsRed2 as a reporter gene. It was PCRed out from the plasmid CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA. A synthetic polyA tail followed by a part of the Hammerhead ribozyme sequence was added on the reverse primer.

Materials and methods

• Miniprep
• Polymerase Chain Reaction

Results

The size of the DsRed2_polyA_HH1 amplicon is 750 bp. The plasmid on the gel is CMVp-dsRed2-Triplex-HHRibo-gRNA1-HDVRibo-pA.