CRISPR Editing ADE2 Gene in S.

cerevisiae
Lena Barko, Melissa Brickett, Abbie Dearinger, Hannah Luchinski, Raegan Mozal
Butler University, Indianapolis, Indiana
Abstract P-ribosyl-PP Results
ADE4
We studied the effectiveness of CRISPR/Cas9 editing by targeting the ADE2 gene in Lane 1 Lane 2 Lane 3
P-ribosylamine
Saccharomyces cerevisiae using a designed sgRNA. Throughout this experiment, we ADE5 Ladder
inserted a sgRNA into an E.coli plasmid which was then transformed into yeast cells. P-ribosylglycinamide
After colonies had formed, we screened for potential mutants by growing them on ADE8
P-ribosylformylglycinamide
suboptimal adenine plates. Additionally, we used a cleaved amplified polymorphism ADE6
screen to determine the success of ssODNA incorporation into the transformed yeast. P-ribosylformylglycinamidine EV gRNA
ADE7
The phenotype screen on the suboptimal adenine plates was unsuccessful; Red clone
P-ribosylamino imidazole
additionally, our restriction digest results were also inconclusive. ADE2 Pigment
P-ribosylamino imidazole carboxylate
ADE1
Figure 4. Proof of plasmid transformation into
P-ribosyl succinocarboxamide aminoimidazole yeast. pML104 yeast containing the URA3 gene
Introduction Adenine Figure 3. Gel electrophoresis of restriction digest analysis
was grown on a uracil (-) agar plate. The
● CRISPR and Cas9, a double stranded endonuclease, use two guide RNA presence of this gene allows it to grow on the
using Kpn1 and BcI1 to prove sgRNA ligated into plate and proves that the plasmid is present in the
sequences to bind to specific invading forgein DNA and destroy it from the Figure 1. Flowchart of the de novo
pML104. Lane 2 shows the empty vector (EV) with two yeast cells as URA3 is not found in wild type
host genome. This process can be manipulated by generating a sgRNA biosynthesis pathway of adenine. Knockout of
Kpn1 bands. Lane 3 shows only one as our sgRNA yeast.
molecule that dictates the endonuclease cut site within a genome. As a result, ADE2 results in accumulation of precursors
replaced the BcI1 cut site and was successfully ligated.
and development of a red pigmentation
Cas9 and the sgRNA can induce DSBs in specific genes researchers intend to
study in any cell. YED Lane 1 2 3 4 5 6 7 8 9 10 11
YPD
● In past experiments, the robust eukaryotic system of S. Cerevisiae has
demonstrated the ability to quickly repair dsDNA breaks via homologous
repair (Laughery et al., 2015)
● The ADE2 gene encodes for AIR-carboxylase which catalyzes a step in the Primer
dimers
purine biosynthetic pathway. As purines make up two of the four nucleotides WT
found in DNA, adenine and guanine, the ADE2 gene can have many impacts
within the yeast cell. Mutations in ADE2 can lead to an accumulation of purine
Figure 5. Phenotypic screen of possibly mutated yeast
precursors in cells. This buildup of purine precursors is what gives the cells the on full nutrient (YPD) or adenine deficient (YED) plates. Figure 7. Genotypic screen of
red color (Gedvilaite and Sasnauskas, 1994; ADE2). Wild type is labeled while other yeast cells could be potential yeast mutants. Yeast
● It is hypothesized that inducing a mutation into the ADE2 gene using CRISPR possible ADE2 mutants. underwent PCR reaction using
technology and HR DNA repair will result in a buildup of purine precursors, designed primers followed by a gel
electrophoresis of EcoRV restriction
demonstrated by an accumulation of a red pigment within the yeast cells.
digest. Ladder is in lane 1, Wild type
● Future experiments could test at at what specific concentrations of adenine the Figure 2. Representation of the pML is lane 2 while potential mutants are
mutant yeast cells begin to alter color. 104 plasmid with specific sites labeled. Figure 6. ssODNA construct used to mutate the ADE2 gene in the yeast cell during HR.
lanes 3-11.

…
Materials and Methods Discussion and Future Directions References
● The phenotypic screen of yeast was unsuccessful as we were unable to identify any red transformed yeast ● ADE2. ADE2 | SGD. (n.d.). Retrieved January 26,
Design gRNAs, primers, Restriction digest on pML104 to insert 2022, from
oligonucleotides, ssODNAs desired sgRNA using SwaI and BcI1 cells when comparing growth on YED to YPD plates (Figure 5). This does not support that the ADE2 gene https://www.yeastgenome.org/locus/S000005654
was mutated due to the lack of purine precursor buildup causing red pigmentation. ● Gedvilaite A, Sasnauskas K. Control of the expression
of the ADE2 gene of the yeast Saccharomyces
● Based on our genotypic screen we cannot confidently conclude that the ADE2 gene knockout was cerevisiae. Curr Genet. 1994 Jun;25(6):475-9. Doi:
Screened E. coli clones via PCR and Purified plasmid to send for Sanger successful. We tested for the presence of EcoRV, a restriction site included in our ssODNA, which would cut 10.1007/BF00351665. PMID: 8082196
if our ssODNA was incorporated into the yeast.The gel electrophoresis indicates 2 bands for our ● Laughery, Marian F., et al. “New Vectors for Simple
restriction digest using Kpn1 and BcI1 Sequencing
and Streamlined CRISPR-Cas9 Genome Editing in
transformed yeast colonies, but there is also a band in the WT lane. Therefore, we cannot be sure that bands
Saccharomyces Cerevisiae.” Yeast, vol. 32, no. 12,
occurred from a ssODNA EcoRV cut site (Figures 6 and 7). 2015, pp. 711–720., https://doi.org/10.1002/yea.3098.
Plasmid transformation into competent Transformation of plasmid and ● When designing our construct, we created 3 sgRNA molecules that target our gene of interest; however, we ● Li SX, Tong YP, Xie XC, Wang QH, Zhou HN, Han Y,
E. coli cells ssODNA into yeast cells and plate chose only one to test. In the future, we could test the other 2 designed sgRNAs to see if they are more Zhang ZY, Gao W, Li SG, Zhang XC, Bi RC.
Octameric structure of the human bifunctional enzyme
successful in knocking out the ADE2 gene in yeast. PAICS in purine biosynthesis. J Mol Biol. 2007 Mar
● Once we have identified a successful and effective knockout of the ADE2 gene in yeast, mutated yeast could 9;366(5):1603-14. doi: 10.1016/j.jmb.2006.12.027.
Genotype screen: CAPS PCR to look Phenotype screen: adenine deficient and be grown on adenine deficient plates and then plated with various adenine concentrations to determine
for EcoRV restriction site cut sufficient plates to scan for red pigment minimum adenine concentration necessary for purine biosynthesis. Acknowledgements
● Continued study of this construct is beneficial because ADE2 has a homologous gene in humans that also
The authors would like to thank Dr. Spears for his guidance
Screening for Mutants: Transformed yeast cells were plated on adenine participates in purine biosynthesis. Studies have shown that cancer cells prefer the purine nucleotides de throughout the experiments and for providing background
optimal and adenine sub-optimal agarose plates. If the ssODNA was novo biosynthesis pathway, making ADE2 an attractive target for anticancer drugs. Further investigating the information to supplement the experiments, Butler
successfully inserted into the yeast cells, the cells grown on the adenine impact of ADE2 in yeast cells could provide essential information relating to the homologous gene in University for the materials, space, and opportunities to
perform the experiments, and the Biology Department for
sub-optimal plates should produce a red pigment. humans and its role in cancer cell proliferation (Li SX et al., 2007). additional help and support.