This method can help reveal the differences between fungi and mammals at the molecular level. Such differences can be leveraged to identify new therapeutic approaches. The main advantage of this technique is that it more efficiently modifies the genome of C.Albicans than classic homologous recombination techniques.
To identify the guide RNA sequence identify an NGG PAM sequence close to where the stop codon will be inserted. Shown here, are all PAM sequences found in the first 100 base pairs of TPK2, a cyclic AMP kinase catalytic subunit. Identify the forward guide primer three sequence.
This sequence will be 20 bases directly upstream of an NGG PAM site and will not contain more than five T's in a row. Left click on the base directly upstream of the NGG and drag the cursor 20 bases. Then, left click on the primer tab to add the primer.
Now, identify the reverse guide primer three sequence, which will be the compliment to the forward guide sequence. Left lick on the base directly upstream of the NGG and drag the cursor 20 bases. For each primer, left click on the primer tab to add the primer.
Right click the primer and select copy primer data. Paste the sequences into a text editing program. Add overhang sequences to forward and reverse guide oligos to facilitate cloning.
Add the nucleotide sequence ATTTG to the five prime end of the forward guide primer three and add G to the three prime end of the forward guide primer three. Finally, at the nucleotide sequence AAAAC to the five prime end of the reverse guide primer three and add C to the three prime end of the reverse guide primer three before purchasing. Dye test the Candida optimized cas9 expression vector by adding pv1524, 10x buffer, bs9b1, and water to 50 micro liters in a 1.5 milliliter tube and incubate at 55 degrees Celsius for 20 minutes.
Cool the digestion mixture to room temperature and spin for 30 seconds at 2348 times G to bring condensation to the bottom of the tube. To phosphatase treat the digested backbone, add one microliter of Calf Intestinal Phosphatase to the digestion mixture. Incubate the reaction at 37 degrees Celsius for one hour before.
Now, phosphorylate and anneal forward guide primer three and reverse guide primer three, by combining them with 10x T4 Ligase Buffer, T4 Polynucleotide Kinase, and water in a PCR tube. Add 10x T4 Ligase Buffer, T4 Polynucleotide Kinase, and Molecular Biology Grade Water to a second PCR tube as a negative control. Incubate the reaction mixtures in a Thermocycler at 37 degrees Celsius for 30 minutes, then at 95 degrees Celsius for 5 minutes.
Cool the mixture at the slowest ramp rate to 16 degrees Celsius to anneal the oligos. Then incubate the annealed oligo mixture at 4 degrees Celsius. Now ligate the annealed oligos into digested PV1524.
In the first PCR tube, add 10x T4 Ligase Buffer, t4 DNA Ligase, annealed oligo mix, digested CIP-treated purified plasmid, and water to a 10 microliter total volume. Similarly prepare a second PCR tube using the negative control mixture instead of the annealed oligo mix. Incubate both tubes in a Thermocycler at 16 degrees Celsius for 30 minutes, then at 65 degrees Celsius for 10 minutes, and finally cool to 25 degrees Celsius.
Following ligation, transform the ligation mixtures into chemically competent cells and then purify and sequence the plasmids as described in the text protocol. To design the repair template, insert a stop codon by left clicking in the gene sequence and adding nucleotides that encode a stop codon and restriction digestion site. Left click 10 bases downstream of where the mutation will be made and drag the cursor 60 bases upstream.
Left lick on the primer tab to add the primer. This will add the repair template forward three. Then left click 10 bases upstream of where the mutation will be made and drag the cursor 60 bases downstream.
Left click on the primer tab to add the primer. This will add the repair template reverse three. To generate the repair template, add repair template forward primer, repair template reverse primer, deoxynucleotide triphosphates, buffer, TACH polymerase, and water to each of the four PCR tubes.
Now perform primer extension by running between 20 and 30 rounds of PCR. To digest the correctly cloned plasmids, add plasmid, 10x Buffer, Bovine Serum Albumin, KPN 1, SAC 1, and water to 40 microliters total volume in a 1.5 milliliter tube. Incubate at 37 degrees Celsius overnight.
Meanwhile, grow an overnight culture of C.albicans SC5314, wild-type prototroph, at 25 degrees Celsius in uridine supplemented YPD. Grow the culture to an optical density at 600 nanometers or OD 600 of less than six. Pellet five OD 600 units of cells per transformation by spinning for five minutes at 2348 times G.Then suspend the pelleted cells in 100 microliters of TE/Lithium Acetate.
Now, to a 1.5 milliliter tube and in order, add the re-suspended cells, boiled and quick-cooled salmon sperm DNA, the plasma digestion, the purified repair template, and plate buffer. Prepare a negative control in the same manner, except to substitute water at a volume equal to that of the transforming DNA. After incubating the transformations overnight, heat chuck the cells by placing them in a 44 degree Celsius water bath for 25 minutes.
Spin for five minutes at 2348 times G in a bench top centrifuge and remove the plate mixture supernatant. Wash once by adding one milliliter of Uridine Supplemented YPD and centrifuge again. After suspending the cells in 0.1 milliliter of Uridine Supplemented YPD, incubate the suspension on a roller drum or shaker at 25 degrees Celsius overnight.
The next day, plate the cells on Uridine Supplemented YPD with 200 micrograms per milliliter of nourseothricin. Colonies will appear in two to five days and should be streaked for single colonies as described in the text protocol. To perform colony PCR, design a forward check primer about 200 base pairs up stream of the restriction site that was introduced, as well as a reverse primer approximately 300 base pairs downstream.
Add 0.3 microliters of forward check primer, 0.3 microliters of reverse check primer, 0.3 microliters of thermostable polymerase, three microliters of dNTP's, three microliters of XTAC buffer, and 23 microliters of water to a tube. Then add 0.25 microliters of a single yeast colony to the mixture using a P10 pipette tip and taking care not to disturb the ager. After amplifying the DNA by PCR, run five microliters of the PCR on the gel to ensure amplification is successful, taking care not to disturb the cell debris pellet at the bottom of the tube.
Representative results for CRISPR mediated genome editing in C.Albicans are shown here. C.Albicans was transformed with guide RNAs and repair templates that target C.Albicans TPK2. An EcoR1 restriction digestion site and stop codons in the repair template disrupt the PAM site and facilitate screening for correct mutants.
Colony PCR followed by restriction digestion quickly distinguishes wild type from edited sequences. While attempting this procedure, it is important to remember to check for multiple copies of the guide RNAs cloned into PV1524. As this will lower genome editing efficiency.
Don't forget, C.Albicans is a BL2 pathogen, and to always where appropriate lab attire and follow all safety procedures while performing this procedure.
