CRISPR-Cas9 gene editing of ADE2 in Saccharomyces cerevisiae

1.0. Introduction

The CRISPR-Cas9 technique is made up of two key components, the Cas9 enzyme and guide RNA (gRNA). It is a new and efficient genetic modified tool that can edit double-strand DNA directly within specific genes through adding, deleting, and altering the sequence (your genome, no date). gRNA is composed of an approximate 20 bases long pre-designed RNA sequence located within a longer RNA scaffold. Cas9 enzyme acts at the specific location to cut the two strands of DNA for further edition. The scaffold part binds to aimed DNA and a pre-designed part of the RNA guides Cas9 to cut at the right place. CRISPR systems were developed from some nature-occurring bacteria responding to invading DNA viruses like an immune system. 

In this experiment, the CRISPR-Cas9 technique was used to cause ADE2 gene mutations in the BY4741 strain of Saccharomyces cerevisiae. Mutation in the ADE2 gene would stop the conversion from P-ribosylation Imidazole to P-ribosylamino imidazolecarboxylate so it would result in the accumulation of P-ribosylamino Imidazole. Accumulation of P-ribosylamino Imidazole intermediate would lead to a pink colour colony phenotype. 

DNA from ADE2 mutant strains was extracted and the mutated region was amplified by PCR. The desired DNA sequencing data was sent and acquired from purified PCR products to see if all the gene editing process successful in this experiment. 

2.0. Aims

Besides interpreting and analyzing DNA sequencing data, this experiment and report are aimed at understanding the mechanism of CRISPR-Cas9, principles behind polymerase chain reaction and primer design also the identification of suitable gRNAs. Moreover, the way to use Zymo Research YeaStar Genomic DNA kit and Qiagen MiniElute PCR Purification kit also plays a crucial role in this practical.

3.0. Results and discussion

3.1.  Transformation of Saccharomyces cerevisiae with pCASgRNA and an HDR DNA fragment

According to Figure 1 and Table 1, the ratio of several distinct pink colonies and distinct white colonies is 303:500, 86:125, and 321:500 respectively in plate 1, plate 2 and in total. 

Overall, in these two plates, most of the yeast cells showed a pink phenotype (54.1%) which means the transformation was successful. In 34.7% of the colonies remained a white phenotype which means the transformation was unsuccessful. 3.5% of the colonies have a mixture of white and pink phenotypes which might suggest the transformation in them hasn’t been completed yet. The rest colonies were indistinct which may be related to the high concentration of yeast cells.