Our protocol explains how to design, build, and analyze yeast gene digital circuits that make use of type two and type five CRISPR dCas systems and the corresponding anti-CRISPR proteins. It's assembly stay because it gathers zero standard procedures to assemble and test synthetic transcriptional networks in services. To begin, prepare a reaction mixture containing 20 to 40 nanograms of DNA template, one microliter of forward primer, one microliter of reverse primer, five microliters of DNTP mix, 0.5 microliters of DNA polymerase, 10 microliters of 5x DNA polymerase reaction buffer, and double distilled water up to a total volume of 50 microliters.
Amplify the DNA sequences using the touchdown PCR program on a thermocycler as described in the manuscript. Isolate the PCR products via gel electrophoresis and dilute the DNA sequences from the agarose gel via a DNA gel extraction kit. Using the Gibson assembly method, insert the purified PCR products into the cut-open shuttle vector by letting in the equimolar DNA mixture at 50 degrees Celsius for one hour.
Construct the dCas12a accepter vector via touchdown PCR and the Gibson assembly method as demonstrated previously. Insert the yeast codon optimized dCas12a proteins into the two newly constructed acceptor vectors via digestion with BamH1 and Xhol1 and ligation with T4 DNA ligase. Similarly, construct the plasmids based on the pRS2403 shuttle vector to express the anti-CRISPR proteins via touchdown PCR and the Gibson assembly method.
To perform microscopy cell detection, place two microliters of the cell solution on a glass slide and cover it with a cover slip. Observe the cells under a fluorescence microscope by turning on the fluorescent light source, microscope, and computer. After writing down the fluorescent light source number, open the microscope software on the computer.
Put the slide on the microscope stage and choose the 40x objective lens while observing the cells under the green light. Move the course focus knob until the contour of yeast cells appears and the fine focus knob to focus the cells. To detect the cells, after closing the microscope field of view switch to the computer screen and click on live.
Wait for three to five seconds. Click on capture to take a picture and save the picture. Next, turn off the computer, microscope, and fluorescent light source.
Switch on the FACS machine 20 minutes before the measurements to warm up the laser and mix 20 microliters of the cell culture with 300 microliters of double distilled water. Run the FACS software on the computer connected to the FACS machine and create a new experiment. Then set the measurement parameters.
Next, select the filter according to the excitation and emission wavelengths of the samples and set the acquisition cell number to 10, 000. Adjust the FITC filter voltage by measuring the intensity of fluorescent beads, ensuring that the relative difference in the intensity of the beads between two consecutive experiments does not exceed 5%Wash the machine with double distilled water for a few seconds to remove any possible excess beads. Measure the sample fluorescence intensity and click on preview while waiting for three to five seconds for sample injection stability.
Finally click on acquire. Measure the beads again at the end of the experiment. After checking whether the relative difference between the two beads measurements exceeds 5%Export the FACS data as FCS files.
Open R studio and load the script BDverse_analysis. R to analyze the FCS files. Set the experiment name as ename, the directory path where the FCS files are stored as dir_d and the result files will be created as dir_r.
Next, set the fluorescence channel. Set the number of samples that were measured. The dimensions of dot plots and the maximal length of the x and y axis for bar plots and box plots.
Choose the grading method by removing the hashtag from the corresponding lines. Select the flowSet object gated corresponding to the chosen gating method and the dot plot resolution, which should be at least equal to 256. Uncomment the two filtering instructions to remove measurements where fluorescence is negative and to remove outliers due to other experiments as described in the manuscript.
Press source and run the script. All the files containing the results from the analysis are created in dir_r. The best activation efficiency of dSpCas9-VP64 was achieved when there were six lexOp target sites on the synthetic promoter.
While for dCas12a-VPR highest activation efficiency when three copies of lexOp were inserted into the synthetic promoter. The activation by CRISPR RNA and single guide RNA located on an integrative plasmid were 1.4 to 2.4, and 1.1 to 1.5 fold higher than when placed on an episomal plasmid. RT-qPCR results showed that an episomal vector produced a much higher level of single guide RNA CRISPR RNA than the integrative vector, irrespective of the expression system.
The activation efficiency of the complex dSpCas9 and scaffold RNA recruiting MCP-VP64 was tested. The scaffold RNA containing a single wild type and F6MS2 hairpin gave a 5.27 and a 4.3 fold activation respectively. However, the combination of the two hairpins resulted in 7.54 fold with the highest activation efficiency.
Four different promoters were used to drive the expression of three kinds of type two anti CRISPR showing that they worked in a dose dependent manner. A strong promoter reduced the fluorescence level to 0.21, 0.11, and 0.13 of its value respectively. The titration curve shows that the circuit behaves like a knot gait with an on to off ratio approaching 2.3.
The type five anti-CRISPR A one reduces the fluorescence expression of both denAS-Cas12a and dLB-Cas12a as activators from 19%to 71%The relation between activators and repressors was studied where the Acr-VA one displayed big fluctuations in its performance when produced by pGAL1 under pTEF1 and genetic CYC1t-pCYC1noTata type five anti-CRISPR-A1 showed some repression on the bare dLB Cas12a only. The FACS machine is frail, therefore it shall be handled carefully. Cell solution should never be too dense and the machine shall be kept clean.
