S. pombe Genome-wide Heterozygous Deletion Mutant Screening Service

Yeast genome-wide knockout library is the world's only fission yeast gene deletion library developed through joint research between the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and the Royal Cancer Institute (Cancer Research UK) (Nature Biotech., 2010). The library is composed of mutant fission yeast (S. pombe) lacking the function of target yeast genes, and targets approximately 98.5% of the genes of the entire fission yeast genome. Gene function-deficient variants were created by inserting an antibiotic resistance gene and a barcode unique to the variant instead of the target gene using the homologous gene recombination method.

GPScreenTM-FAST balances the growth of all variants that make up the library and then pools them for service.

Drug-induced haploinsufficiency (DIH) is a diploid yeast mutant with a defect in the function of one copy of the gene. The remaining normal copy of the gene does not produce a sufficient amount of protein for normal function, and part of the function is lost, causing the yeast to malfunction. This is a phenomenon in which strains become more sensitive to drug treatment.

Because strains showing this increased sensitivity indicate that the decreased function of these genes is associated with the drug's mechanism of action, which has a significant impact on cell survival or function, it is likely that the missing gene is related to the drug's mechanism of action.

In general, a DNA barcode refers to a specific DNA sequence used to identify a biological species. In a similar sense, the DNA barcode in the yeast genome-wide knockout library refers to the unique DNA sequences of the target gene knockout strains that make up the library. Therefore, DNA barcodes can be used to identify knockout strains using DNA sequencing.

In GPScreenTM-FAST, the growth sensitivity of each knockout strain constituting the pool due to drug treatment is measured by quantifying the DNA barcode of each knockout strain using NGS (Next Generation Sequencing) technology. Analyze drug sensitivity based on quantified DNA barcode values and search for genes related to drug action mechanisms.

IC50 (half-maximal inhibitory concentration), a term commonly used in new drug development, refers to the concentration of a drug that inhibits biological processes by 50%. In a similar sense, GI50 (growth inhibition) in GPScreenTM-FAST refers to the drug concentration when cytotoxicity caused by drug treatment inhibits yeast growth by 50% compared to yeast growth under normal conditions.

In order to proceed with the GPScreenTM-FAST service, which is a drug target identification service, a service that finds the GI50 of the drug in question must be performed first [Primary Test Service in Wild Type S.pombe (GPS-00)]. GI50 screening of drugs is performed on wild-type S.pombe (SP286) rather than a yeast genome-wide knockout library pool, and is usually confirmed at a concentration within 1 mM of the GI50. Determining the GI50 of a drug may be difficult if the drug is not cytotoxic in yeast cells.

Yeast, which is known to have a metabolome similar to that of humans, is well known as a microbial model for human cell research, and human cell research using yeast is being continuously conducted. In particular, among yeasts, S.pombe has a preserved metabolic pathway similar to that of humans, making it more advantageous for human research than other yeast species. In some cases, key regulatory proteins such as Cdc25 and Cdk1 of the human cell cycle were discovered for the first time in a study using S.pombe. And until recently, yeast-based genome-wide phenotypic screen (phenotypic analysis) was reported as a promising technology that can be used in human genome research (2023, Sci. Adv. 9:eadg5702).

There are several advantages to using the yeast genome-wide knockout library produced using this excellent human research model yeast (S.pombe) for drug target identification research.

• In yeast (S.pombe), genome-wide level drug action gene network analysis is possible. Due to the large number of genes and genetic variants in human cells (approximately 20,000), analyzing the drug response gene network at the genome-wide level in human cells is a very complex task. On the other hand, yeast (S.pombe), a human research model, shares many core human genes out of a total of about 5,000 genes, and in particular, more than 1,000 genes are known to be human disease-related genes. In other words, because S.pombe is a compressed genome in which major human genes are preserved, it is possible to analyze drug response molecular mechanisms at a genome-wide level targeting only relatively few but major genes.
• In yeast (S.pombe), various drugs can be analyzed quickly and inexpensively. Compared to animal cells, research using yeast cells allows the identification of drug targets at a low cost because the research costs, including facilities and culture media required for management, are relatively low. Additionally, since the division cycle time of S.pombe is only 2 to 4 hours, it has the advantage of being able to conduct drug target identification research faster than animal cells, which have a division cycle time of one day. And unlike animal cells, which require drug modification depending on the drug target identification method, any drug can be analyzed in yeast as long as the drug has cytotoxic properties.