Although the author's previous screening strategy for identifying human gene substitution variants in yeast genes was successful, the strategy was cumbersome and only identified two repressors (Kachroo et al., 2015). This technology requires manual isolation of bacterial colonies, followed byTetradSeparation and identification of inhibitors require manual screening of hundreds of yeast colonies. Therefore, the author developed a new process to screen inhibitory plasmids in an automated high-throughput manner (Figure 1a).
The author specifically focuses on screening for suppressor mutations in non complementary human β 2c (HsPSMB7) protein, which is a constitutive expression of proteasome subunits. This method can achieve large-scale and error free screening in a significantly shortened time. The experiment was conducted in the form of a 96 well plate and synthesized using a genetic array（SGA）Alternatively, the MM selection method can be used to isolate haploid specific mutants, eliminating the need for tetrad isolation (Pan et al., 2004); Kuzmin et al., 2016). By using plasmid dependent assays with 5-FOA selection, it was confirmed that complementary effects are associated with mutated human genes on URA3 based plasmids. Using this strategy, the author successfully obtained multiple replaceable inhibitory mutations in the HsPSMB7 gene. Throughout the paper, the author consistently uses terms such as "functional substitution," "replaceability," or "replaceability" to refer to the ability of human genes or their variants to compensate for the function of their yeast homologous genes. This screening process is developed based on multiple considerations. The wild-type human PSMB7 (β 2c) cannot replace the homologous yeast β 2 gene ScPUP1, as evidenced by the lethal phenotype observed when selectively removing yeast genes (Figure 1b, Supplementary Figure 1b) (Kahlo et al., 2015). In contrast, if human genes (or their variants) successfully replace the function of host genes, the strain will be able to grow and serve as a simple detection indicator for functional substitution. The PCR strategy with high error rate generated an HsPSMB7 mutation gene library in yeast expression vectors labeled with URA3, with an average of 1 to 4 mutations per gene (Figure 1a).
To determine whether any mutated PSMB7 allele can compensate for the lethality caused by deletion of homologous yeast genes, the authors transformed the mutation gene library into the yeast diploid HetKO PUP1/pup1 Δ: kanMX strain (Pan et al., 2004). The transformation scheme was extended to obtain thousands of isolated yeast colonies, each carrying a different PSMB7 mutant gene plasmid (Figure 1b). Thousands of colonies were automatically selected using QPix 460 and inoculated into pre sporulation GNA medium in a 96 well formatG418The selection method is used to screen Pup1 Δ with kanMX markers. After spore formation, viable Pup1 Δ:: kanMX haploid yeast spores carrying different human PSMB7 alleles can be selected in MM medium (excluding Leu, Arg, His, Ura, and CAN) in the presence of G418 (Figure 1c, bottom panel). As an internal control for spore formation efficiency, the authors also tested the growth of wild-type PUP1 haploid spores on MM medium (without G418) (Figure 1c, top panel). The screening results showed that 19 colonies grown on MM+G418 medium (representative image in Figure 1d) may carry complementary human PSMB7 variants. Then, these haploid inhibitory mutants were tested to determine whether the inhibition was caused by carrying human mutant genes in the plasmid. Test the plasmid dependence of yeast cells based on the lack of growth of 5-FOA, which inhibits the URA3 gene. Among these 19 inhibitory mutants, 7 were unable to survive on 5-FOA medium, indicating that human gene variants in these strains are essential for their survival (Figure 1d).
Figure 1: A high-throughput automated process for screening human gene inhibitors with substitutability in yeast. a) The workflow demonstrates the process of generating and screening a human gene mutation library through mismatch PCR (0-4 mutations per kilobase pair). The mutation pool was cloned into expression vectors (CEN6, URA3) and transformed into yeast heterozygous diploid knockout PUP1/pup1 Δ:: kanMX strain. b) The transformation scale of the mutation library was expanded by 4 times, and the mixture was coated on QTray. Single colonies were selected using the QPix 460 colony selection robot (up to 1000 colonies) and spotted on pre spore formation medium rich in GNA, followed by spore formation in a 96 well plate format. c) Each spore forming mixture was spotted on MM medium containing G418, indicating the condition of "yeast gene deletion and human gene presence", for screening human gene variants with substitutability. Alternatively, a mixture of yeast genes present or absent and human genes present without the addition of G418 spores is allowed to grow wild-type haploid yeast cells as a control for spore formation efficiency. For simplicity, we will refer to these conditions as "containing (+) G418" to indicate "yeast gene deletion" condition, and "not containing (-) G418" to indicate "yeast gene presence" condition. Possible inhibitors may manifest as spots growing on MM medium containing G418, similar to the growth conditions under MM medium without G418. d) The colonies grown on MM medium containing G418 (yeast gene deletion and human gene presence conditions) were further analyzed by plasmid dependent assays using 5-fluorouracil (5-FOA) selection targeting the URA3 plasmid. Representative examples show that yeast strains carrying human gene repressors do not grow on 5-FOA medium, indicating plasmid dependence (repressor 1, top image), or grow on 5-FOA medium, indicating plasmid independence (repressor 2, bottom image). Finally, each plasmid carrying human gene repressors was purified and retested to confirm functional substitutability and plasmid dependence, followed by Sanger sequencing to identify mutations in human genes.
