Targeting QTLs using the CRISPR/Cas9 system in yeast

Background and objectives: There is still limited knowledge about how phenotypes arise from genetic loci. It is especially difficult to understand complex traits that are affected by several both genetic and environmental factors. In order to understand these traits in more detail quantitative trait loci (QTL) and their effect on a phenotype can be studied. However, so far it has been difficult to determine which of the many loci within a QTL actually have a causal effect on the phenotype. Therefore, in this Master's thesis the aim was to establish a QTL replacement protocol using the CRISPR/Cas9 genetic engineering in order to study the effects of individual variant within a QTL interval in detail. Methods: A protocol replacing single nucleotide long QTLs in the yeast genome was designed. Three single nucleotide polymorphism (SNPs) were identified within the TDP1 gene in the yeast genome. These SNPs were all located within the interval of a growth QTL, thus potentially affecting the growth of the cell. In addition, two protocols, same locus tagging and dual targeting, were designed for the selection of the correctly edited cells when using the CRISPR/Cas9 system and the same locus tagging was also partly tested. As a model organism S. cerevisiae was used since it is a rather simple eukaryotic organism that has already been used a lot in CRISPR/Cas9 experiments. Results: As a result of the QTL replacement experiment correct mutations appeared only when the sequence quality was poor. Therefore, it is very likely that these mutations did not result from the successful CRISPR/Cas9 experiment. In the same locus tagging protocol the selection for Cas9 using overnight-lasting geneticin exposure was found to increase the amount of correctly edited cells. Conclusions: Despite the lack of the target modifications this study is able to give new information about the limitations of the CRISPR/Cas9 system and indeed, several ways for improving the protocol were identified. Despite its limitations the CRISPR/Cas9 system can definitely be seen as one of the most promising methods for genetic engineering