This protocol demonstrates a novel method for Multiplex Genome Editing of Saccharomyces cerevisiae by introducing multiple expression cassettes on different loci in a single transformation step. This cloning free plasmid assembly approach for expressing a single crRNA array, leads to a rapid, efficient, and flexible genome editing protocol. We will engineer wild type yeast cells, to produce carotenoids by introducing free heterologous genes on free genomic loci.
The single crRNA array is composed of free individual crRNAs expressed from RNA polymerase free promoter. Cas12a processes in vivo the crRNA array into individual crRNAs. The crRNAs guide Cas12a to the target loci on the genomic DNA where Cas12a introduces double strand breaks.
Downward DNA fragments recombine for in vivo recombination and integrate into genomic DNA. Demonstrating the protocol will be Klaudia Ciurkot, a PhD student. Jeffery van Wijk, an Associate Scientist.
And Brenda Vonk, a Senior Associate Scientist from our laboratory. After obtaining a single crRNA array for Multiplex Genome Editing experiments of synthetic DNA, prepare the PCR amplification mix, and load the array into the thermocycler for amplification. To analyze the PCR products by electrophoresis, run the samples on a 0.8%agarose gel at 5 V/cm for 40 minutes.
Load the DNA ladder with the DNA fragments ranging from 100 10000 base pairs Then purify the PCR products using a PCR purification kit according to the instructions of the manufacturer. For yeast transformation, begin by pre-culturing wild type yeast in a 100 mL flask containing 20 mL of yeast extract peptone dextrose or YEPD medium. For an overnight incubation, at 30 degrees Celsius, and 250 rotations per minute The next morning, inoculate 20 mL of fresh YEPD medium with a calculated volume of overnight pre-culture yeast.
Place the culture in a shaking incubator until an optical density of 1.0, measured at 600 nanometers is reached. Harvest the yeast cells by centrifugation. Wash the yeast cell pellet in 20 mL of room temperature demineralized water, followed by an additional centrifugation.
Resuspend the pellet in 100 microliters of lithium acetate Tris-EDTA solution, and transfer the yeast to a microcentrifuge tube. Mix five microliters of single-stranded carrier DNA and one microgram of plasmid pCSN067 with the yeast cell suspension. Then mix 600 microliters of polyethylene glycol LiAc-TE solution with the sample.
Incubate the yeast cells and plasmid for 30 minutes at 30 degrees Celsius and 450 prm in a table top heat block. At the end of the incubation, mix 70 mL of dimethyl sulfoxide with the transformation solution. Heat shock the mixture for 15 minutes in a 42 degrees Celsius water bath.
Transfer the mixture to a 15 mL round bottom tube. Add 10 mL of YEPD to the tube for an overnight incubation at 30 degrees Celsius and 250 rpm. The next morning, collect the transformed cells by centrifugation, and aspirate all but the final 200 microliters of the supernatant.
Resuspend the transformed yeast cell pellet in the remaining supernatant. Plate 150 microliters of the transformation mix, and 150 microliters of a 20 times dilution of the remaining 50 microliters of transformation mix in YEPD, on YEPD agar plates, supplemented with 0.2 grams per liter of G418. After 48 to 72 hours at 30 degrees Celsius, pick a single transformant for re-streaking on a new YEPD agar plate supplemented with 0.2 g/L of G418.
For the second transformation, grow the culture to an optical density at 600 nanometers of 1.0, as just demonstrated. And add 20 microliters of cells to a microcentrifuge tube. After centrifugation, resuspend the cell pellet in 100 microliters of LiAc-TE solution, and add ssDNA.
In a separate tube, combine one microgram of the single crRNA array, one microgram of the linearized recipient plasmid for the crRNA array, one microgram of each donor DNA, and one microgram of each flanking region. Then transfer the DNA mixture to the tube of competent yeast cells and complete the second transformation as demonstrated for the first transformation. The next morning, collect the cells by centrifugation and resuspend the pellet in a small aliquot of supernatant.
Then plate 150 microliters of the transformation mix, and 150 microliters of a 20 times dilution of the transformation mix in YEPD medium, onto YEPD agar plates supplemented with 0.2 g/L G418, and 0.2 g/L nourseothricin After 48 to 72 hours, at 30 degrees Celsius, pick a single colored transformant for re-streaking on a new YEPD plate to obtain single colored colonies. To determine the genome editing efficiency, count the number of colored and white colonies on the transformation plates, and divide the number of colored colonies by the total number of colonies. To create yeast pixel art, inoculate individual 500 mL shake flasks containing 100 mL of YEPD medium with three differently colored carotenoid producing S.cerevisiae strains, and a wild type CEN.
PK113-7D strain. Incubate the cultures overnight at 30 degrees Celsius with shaking at 250 rpm. Transfer 0.5 mL of each overnight culture to individual tubes containing 0.5 mL of sterile, non-ionic density gradient medium per tube, and briefly mix the solutions by vortexing.
Transfer the cell suspensions to individual, qualified reservoir 2 by 3 wells. Use a csv file with the fluid calibration setting 6RES_AQ_GPSA2 on an acoustic liquid handler instrument to spot 25 nanoliters of each S.cerevisiae strain from the appropriate qualified reservoir source plate onto a 6144 well destination microplate containing 50 mL of YEPD agar. When all of the wells have been spotted, incubate the microplate at 30 degress Celsius for 48 hours.
To intensify the colors of the strains, store the agar plates at 4 degrees Celsius for at least 72 hours. A picture of Rosalind Franklin has appeared on the plate. As demonstrated, promoter, open reading frame, terminator, and two contiguous 50 bp connector sequences can be assembled into an expression cassette via a Golden Gate cloning reaction, and verified by PCR.
After amplification of the single crRNA array by PCR, the recipient plasmid for the single crRNA array is linearized as confirmed by electrophoresis. The efficiency of the Multiplex Genome Editing can be calculated by the percentage of colored colonies after the transformation, and by the results of PCR analyses that confirm the integration of donor DNA at the intended genomic DNA locations. For example, starting from a black and white picture of Rosalind Franklin, a four color picture and spotting list was created that was then used to spot the four different yeast strains on an agar microplate via an acoustic liquid handler, as demonstrated, resulting in a high resolution yeast painting of the scientist.
For this transformation, use freshly prepared solutions and DNA fragments of a high quality. This method can also be applied for multiplex introduction of defined deletions and point mutations into genomic DNA. In addition, CRISPR regulation experiments can be performed.
This method can be modified to introduce more than free crRNAs within an array to increase the number of simultaneous modifications that can be made.
