Yeast Library Research Articles

Applications of CRISPR/Cas gene-editing technology in yeast and fungi.

Genome editing technology has progressed rapidly in recent years. Although traditional gene-editing methods, including homologous recombination, zinc finger endonucleases, and transcription activator-like effector nucleases, have substantial implications for research in genetics and molecular biology, but they have remarkable limitations, including their low efficiency, high error rate, and complex design. A new gene-editing technology, the CRISPR/Cas system, was developed based on studies of archaeal and bacterial immune responses to viruses. Owing to its high target efficiency, simple primer design, and wide applications, the CRISPR/Cas system, whose developers were awarded the Nobel Prize in Chemistry in 2020, has become the dominant genomic editing technology in academia and the pharmaceutical industry. Here, we briefly introduce the CRISPR/Cas system and its main applications for genome engineering, metabolic engineering, and transcriptional regulation in yeast, filamentous fungi, and macrofungi. The polygene and polyploid editing, construction of yeast chromosomes, yeast library creation, regulation of metabolic pathways, and CRISPR activation/CRISPR interference systems are mainly summarized and discussed. The potential applications for the treatment of fungal infections and the further transformation and application of the CRISPR/Cas system in fungi are also proposed and discussed.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

An engineered CD81-based combinatorial library for selecting recombinant binders to cell surface proteins: Laminin binding CD81 enhances cellular uptake of extracellular vesicles.

The research of extracellular vesicles (EVs) has boomed in the last decade, with the promise of them functioning as target‐directed drug delivery vehicles, able to modulate proliferation, migration, differentiation, and other properties of the recipient cell that are vital for health of the host organism. To enhance the ability of their targeted delivery, we employed an intrinsically overrepresented protein, CD81, to serve for recognition of the desired target antigen. Yeast libraries displaying mutant variants of the large extracellular loop of CD81 have been selected for binders to human placental laminin as an example target. Their specific interaction with laminin was confirmed in a mammalian display system. Derived sequences were reformatted to full‐length CD81 and expressed in EVs produced by HeLa cells. These EVs were examined for the presence of the recombinant protein and were shown to exhibit an enhanced uptake into laminin‐secreting mammalian cell lines. For the best candidate, the specificity of antigen interaction was demonstrated with a competition experiment. To our knowledge, this is the first example of harnessing an EV membrane protein as mediator of de novo target antigen recognition via in vitro molecular evolution, opening horizons to a broad range of applications in various therapeutic settings.

Construction of Yeast One-Hybird Library for Screening of Eugenol Synthase Gene Bait vectors in Rosa chinensis

Roses are cultivated as ornamental plants in gardens, cut flowers and potted flowers, but also serviced in the perfume industry. Rose flowers product different volatile compounds including eugenol, being floral attractant for pollinators and used as an important composition of essential oil. Here, the eugenol synthase gene (RcEGS1) promoter was cloned from R. chinensis 'Old Blush'. The sequences of RcEGS1 promoter were 1776bp, contained the conserved elements, and predicted to be with several cis-regulatory elements involved in secondary metabolism of plants. Yeast one-hybrid library of R. chinensis 'Old Blush' at flower opening was constructed with the three-frame reading. The library capacity was more than 1.12×107CFU. The length of fragments inserted was 1 Kb to 3 Kb, which is fully meeting the demand of selecting the yeast library. The bait yeast vectors with cis-transcription elements MBS-box, G-box and one fragment bait yeast vectors were constructed, respectively. Furthermore, the transcript factors R2R3-type MYB and bHLH3 were screened using the yeast one-hybrid system. The results suggested the two TFs might play an important role in the pathway of volatile biosynthesis in roses.

A high-throughput screening method forthe discovery of Saccharomyces and non-Saccharomyces yeasts with potential in the brewing industry.

Both Saccharomyces and non-Saccharomyces yeast strains are of great importance for the fermentation industry, especially with the flourishing of craft breweries, which are driving current innovations. Non-conventional yeasts can produce novel beverages with attractive characteristics such as flavour, texture, and reduced alcohol content; however, they have been poorly explored. A new method for screening the fitness of conventional and non-conventional yeast libraries utilising robotic platforms and solidified media representing industrial conditions is proposed. As proof of concept, a library formed of 6 conventional and 17 non-conventional yeast strains was distributed in 96, 384 and 1536 arrays onto a YPD agar medium. Following this, the library was replicated in different conditions mimicking beer and cider fermentation conditions. The colony size was monitored over time, and fitness values measured in maximum pixels/h and maximum biomass were calculated. Significant differences in growth were observed in between the different strains and conditions. As examples, Candida milleri Y-7245 displayed good performance in wort conditions, and Kazachstania yakushimaensis Y-48837 stood out for its performance in apple juice. The method is proposed to be used as a pre-screening step when studying vast yeast libraries. This would enable interested parties to discover potential hits for further study at a low initial cost. Furthermore, this method can be used in other applications where the desired screening media can be solidified.

Ethylene-activated MdPUB24 mediates ubiquitination of MdBEL7 to promote chlorophyll degradation in apple fruit.

Chlorophyll (Chl) degradation is a natural phenomenon that occurs during ripening in many fleshy fruit species, and also during fruit storage. The plant hormone ethylene is a key factor in promoting Chl degradation during fruit storage, but the mechanisms involved in this induction are largely unknown. In this study, an apple (Malus domestica) BEL1-LIKE HOMEODOMAIN transcription factor 7 (MdBEL7), potentially functioning as a transcriptional repressor of the Chl catabolic genes (CCGs), including MdCLH, MdPPH2 and MdRCCR2, was identified as a partner of the ethylene-activated U-box type E3 ubiquitin ligase MdPUB24 in a yeast library screen. Yeast-two-hybrid, co-immunoprecipitation and luciferase complementation imaging assays were then used to verify the interaction between MdBEL7 and MdPUB24. In vitro and invivo ubiquitination experiments revealed that MdPUB24 functions as an E3 ubiquitin ligase to ubiquitinate MdBEL7, thereby causing its degradation through the 26S proteasome pathway. Transient overexpression of MdPUB24 in apple fruit led to a decrease in MdBEL7 abundance and increased expression of CCG genes, including MdCLH, MdPPH2 and MdRCCR2, as well as greater Chl degradation. Taken together, the data indicated that an ethylene-activated U-box type E3 ubiquitin ligase MdPUB24 directly interacts with and ubiquitinates MdBEL7. Consequent degradation of MdBEL7 results in enhanced expression of MdCLH, MdPPH2 and MdRCCR2, and thus Chl degradation during apple fruit storage. Our results reveal that an ethylene-MdPUB24-MdBEL7 module regulates Chl degradation by post-translational modification during apple fruit storage.

A genome‐scale yeast library with inducible expression of individual genes

The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single‐gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high‐throughput genetics. We characterized YETI using growth phenotyping and BAR‐seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter‐perturbing screens, including CRISPRi. We engineered a second expression system, Z3EB42, that gives lower expression than Z3EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.

Oncohistone mutations enhance chromatin remodeling and alter cell fates.

Whole genome sequencing data mining efforts have revealed numerous histone mutations in a wide range of cancer types. These occur in all four core histones in both the tail and globular domains and remain largely uncharacterized. Here we used two high-throughput approaches, a DNA-barcoded mononucleosome library and a humanized yeast library, to profile the biochemical and cellular effects of these mutations. We identified cancer-associated mutations in the histone globular domains that enhance fundamental chromatin remodeling processes, histone exchange and nucleosome sliding, and are lethal in yeast. In mammalian cells, these mutations upregulate cancer-associated gene pathways and inhibit cellular differentiation by altering expression of lineage-specific transcription factors. This work represents a comprehensive functional analysis of the histone mutational landscape in human cancers and leads to a model in which histone mutations that perturb nucleosome remodeling may contribute to disease development and/or progression.

Mapping antibody escape in SARS-CoV-2

Coronavirus Several antibodies are in use or under development as therapies to treat COVID-19. As new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants emerge, it is important to predict whether they will remain susceptible to antibody treatment. Starr et al. used a yeast library that covers all mutations to the SARS-CoV-2 receptor-binding domain that do not strongly disrupt binding to the host receptor (ACE2) and mapped how these mutations affect binding to three leading anti–SARS-CoV-2 antibodies. The maps identify mutations that escape antibody binding, including a single mutation that escapes both antibodies in the Regeneron antibody cocktail. Many of the mutations that escape single antibodies are circulating in the human population. Science , this issue p. [850][1] [1]: /lookup/doi/10.1126/science.abf9302

Function-focused protein design

Protein Design De novo design allows the creation of new types of stable folds in proteins; however, building in successful function remains challenging. Yang et al. describe a bottom-up approach in which they conceived structures around functional motifs and then used targeted screening to optimize their composition. Based on thousands of blueprints, they screened yeast libraries in which most residues were restricted to those found most frequently in the designs, but critical core residues were allowed limited variability constrained by these designs. This approach enabled the authors to discover five folds that accommodated four different binding motifs, including a protein that displays two motifs. They then built proteins that are functional as biosensors of specific antibody epitopes and others that act as ligands to regulate synthetic receptors in mammalian cells. Nat. Chem. Biol. 10.1038/s41589-020-00699-x (2020).

Prospective mapping of viral mutations that escape antibodies used to treat COVID-19.

Antibodies are a potential therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the risk of the virus evolving to escape them remains unclear. Here we map how all mutations to the receptor binding domain (RBD) of SARS-CoV-2 affect binding by the antibodies in the REGN-COV2 cocktail and the antibody LY-CoV016. These complete maps uncover a single amino acid mutation that fully escapes the REGN-COV2 cocktail, which consists of two antibodies, REGN10933 and REGN10987, targeting distinct structural epitopes. The maps also identify viral mutations that are selected in a persistently infected patient treated with REGN-COV2 and during in vitro viral escape selections. Finally, the maps reveal that mutations escaping the individual antibodies are already present in circulating SARS-CoV-2 strains. These complete escape maps enable interpretation of the consequences of mutations observed during viral surveillance.

Yeast Two-hybrid cDNA Library Constructed from Vitis davidii Fex Pericarps Infected by Grape Ripe Rot Pathogen, Colletotrichum viniferum

Objective A yeast two-hybrid cDNA library on Colletotrichum viniferum-infected pericarps of the wild spine grape (Vitis davidii Foex), a cultivar highly resistant to the serious grape ripe rot in southern China, was constructed to facilitate studies on the molecular mechanism of the disease resistance in the plants. Method The total RNA was extracted from the pericarps of V. davidii accession, Fu'an, 1, 3, and 7d after C. viniferum inoculation and reverse-transcribed into cDNA using the SMART method. The cDNA was purified by an assay kit into a double stranded cDNA to be digested. Then, the short fragments were removed to obtain a high-quality cDNA for the subsequent cloning into a pGADT7 3-frame plasmid vector and purifying for the establishment of a primary cDNA library. After amplification, the plasmid was extracted and transformed into Yeast Y187 to produce the amplified library for final identification. Result The primary cDNA library had a capacity of approximately 5.2×106 cfu with a recombination rate of approximately 97.92% and the inserted fragments of good polymorphism in the lengths ranging from 400 to 2 000 bp. The harvested Y187 yeast library titer was about 6.0×107 cfu·mL−1. Conclusion The constructed cDNA library from C. viniferum-infected grape pericarps was adequate for yeast two-hybrid screening. It would materially aid the studies on the interacting proteins in grape plants infected by the pathogen.

OsYUC11-mediated auxin biosynthesis is essential for endosperm development of rice.

Auxin is a phytohormone essential for plant development. However, our understanding of auxin-regulated endosperm development remains limited. Here, we described rice YUCCA (YUC) flavin-containing monooxygenase encoding gene OsYUC11 as a key contributor to auxin biosynthesis in rice (Oryza sativa) endosperm. Grain filling or storage product accumulation was halted by mutation of OsYUC11, but the deficiencies could be recovered by the exogenous application of auxin. A rice transcription factor (TF) yeast library was screened, and 41 TFs that potentially bind to the OsYUC11 promoter were identified, of which OsNF-YB1, a member of the nuclear factor Y family, is predominantly expressed in the endosperm. Both osyuc11 and osnf-yb1 mutants exhibited reduced seed size and increased chalkiness, accompanied by a reduction in indole-3-acetic acid biosynthesis. OsNF-YB1 can bind the OsYUC11 promoter to induce gene expression in vivo. We also found that OsYUC11 was a dynamically imprinted gene that predominantly expressed the paternal allele in the endosperm up to 10 d after fertilization (DAF) but then became a non-imprinted gene at 15 DAF. A functional maternal allele of OsYUC11 was able to recover the paternal defects of this gene. Overall, the findings indicate that OsYUC11-mediated auxin biosynthesis is essential for endosperm development in rice.

BaSDAS: a web-based pooled CRISPR-Cas9 knockout screening data analysis system.

We developed the BaSDAS (Barcode-Seq Data Analysis System), a GUI-based pooled knockout screening data analysis system, to facilitate the analysis of pooled knockout screen data easily and effectively by researchers with limited bioinformatics skills. The BaSDAS supports the analysis of various pooled screening libraries, including yeast, human, and mouse libraries, and provides many useful statistical and visualization functions with a user-friendly web interface for convenience. We expect that BaSDAS will be a useful tool for the analysis of genome-wide screening data and will support the development of novel drugs based on functional genomics information.

Abstract 598: Integrated computational and experimental design of a monobody targeting PDL1

Abstract Monobodies (Mb) are small, synthetic antibodies based on the human fibronectin type III domain that exhibit high thermodynamic stability without disulfide bonds. To explore suitability of these domains as cancer-targeting molecules, we apply computational and experimental methods to develop a Mb that targets programmed-death-ligand 1 (PDL1). As some tumors overexpress PDL1 to inhibit T-cell responses, a PDL1-binding Mb could be used to promote successful immunotherapy. More broadly, computational methods applied to developing a PDL1-binding Mb can also be applied to developing new Mbs that target cancer antigens. Starting from the G9 Mb (PDB: 1ttg), we grafted the CDR3 loop of a known PDL1-binder (PDB: 5jds) into the FG loop of the Mb. This design was displayed on the surface of yeast and shown to bind PDL1 better than the G9 Mb, though not as well as a high affinity variant of PD1, the cognate receptor of PDL1. Using molecular dynamics simulations, we evaluated structural features that are unlikely to contribute to PDL1-binding. Using this approach, we selected amino acid positions for site-saturated mutagenesis and constructed a yeast library displaying Mb isoforms. Through iterative rounds of selection, we identified a Mb isoform with further improved PDL1-binding. Citation Format: Reed E. Harrison, Chi-Wei Man, Yingxiao Wang. Integrated computational and experimental design of a monobody targeting PDL1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 598.

A Leymus chinensis histidine-rich Ca2+-binding protein binds Ca2+/Zn2+ and suppresses abscisic acid signaling in Arabidopsis

Intracellular Ca2+ plays an essential role in plant cellular sensing of various environmental stress signals by modulating the activity of Ca2+-binding proteins. Leymus chinensis is a dominant forage grass widely distributed in the Eurasian Steppe that is well adapted to drought and salty soils common in the region. Through transcript profiling of L. chinensis roots, we identified a transcript predicted to encode histidine-rich calcium-binding protein (HRC), a protein recently characterized in wheat. L. chinensis HRC (LcH RC) localized in the nucleus, as demonstrated using a transient gene expression method that we developed for this species. Different regions of LcHRC showed affinity for either Ca2+ or Zn2+, but not Mg2+ and Mn2+. Arabidopsis thaliana seedlings heterologously overexpressing LcHRC showed greater sensitivity to abscisic acid (ABA), along with decreased expression of some ABA-induced marker genes, but no increase in ABA content. Screening a Arabidopsis cDNA yeast library identified a Tudor/PWWP/MBT-domain-containing protein (AtPWWP3) that interacts with LcHRC. AtPWWP3 also localized in the nucleus and is predicted to mediate gene expression by modifying histone deacetylation. Based on these results, we propose a functional model of LcHRC action.

Identifying Prospective Genes That Function During DNA Repair in the Bdelloid Rotifer Adineta vaga

The bdelloid rotifer Adineta vaga is a microinvertebrate with a highly efficient DNA repair system that allows individuals to recover from DNA damage caused by desiccation or ionizing radiation. The mechanism behind this remarkable DNA repair in A. vaga is not yet understood. Previous transcriptome sequencing in our lab has identified over 250 genes that are upregulated in response to DNA damage induced by ionizing radiation. These upregulated genes are the strongest candidates for having a role in DNA repair. The objective of this project is to characterize the functions of candidate genes involved in DNA repair in bdelloids and, thereby, establish potential pathways that comprise this mechanism. Three approaches were used to begin to investigate the functions of these candidate DNA repair genes in bdelloids. First, we used real‐time PCR to quantify gene expression of several candidate DNA repair genes during recovery from desiccation. Genes that were upregulated relative to the non‐desiccated control were identified as most likely to have a role in DNA repair as opposed to radiation resistance. Second, we constructed a yeast library of A. vaga proteins for use in yeast two‐hybrid screens against candidate DNA repair proteins. To produce the library, cDNAs representing the full complement of A. vaga proteins were cloned into plasmids that were then transformed into yeast. Yeast strains that each expressed a different candidate DNA repair protein would then be mated with the library to screen for protein‐protein interactions, which would provide evidence for related cellular functions. Third, we developed the molecular tools for using CRISPR/Cas9 genome editing to inactivate candidate genes in A. vaga. In vivo gene knockouts would provide mutant rotifers that could be studied to assess the roles of these genes in DNA repair. Single guide RNAs (sgRNAs) for several genes were created and incubated with the Cas9 enzyme to form sgRNA‐Cas9 complexes. These sgRNA‐Cas9 complexes were shown to successfully cleave their target gene sequences in vitro. To test this method in vivo, these sgRNA‐Cas9 complexes were transfected into A. vaga embryos via electroporation. Genomic DNA was purified from treated rotifers and used in T7 endonuclease I assays to identify subsequent mutations in the target genes. Once perfected, these methods of investigating the roles of candidate genes stand to elucidate the mechanism behind the extraordinary DNA repair in A. vaga.Support or Funding InformationFunding was provided by a Summer Undergraduate Research Fellowship from the Arkansas Department of Higher Education to JTW, and a Hendrix College Faculty Project Grant to AMS.

Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries.

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

Multi-functional genome-wide CRISPR system for high throughput genotype\u2013phenotype mapping

Genome-scale engineering is an indispensable tool to understand genome functions due to our limited knowledge of cellular networks. Unfortunately, most existing methods for genome-wide genotype–phenotype mapping are limited to a single mode of genomic alteration, i.e. overexpression, repression, or deletion. Here we report a multi-functional genome-wide CRISPR (MAGIC) system to precisely control the expression level of defined genes to desired levels throughout the whole genome. By combining the tri-functional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported. The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels. MAGIC represents a powerful synthetic biology tool to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.

Development of a new high-throughput method for screening large yeast libraries for use in the beverage industry

High-throughput techniques have become crucial for the discovery of new strains suitable for the beverage industry. These approaches allow assessment of critical traits for fermented beverages such as; stress tolerance, growth, viability, vitality and aromatic compound production in a fast and effective manner. The growth rate of 24 Saccharomyces and non-Saccharomyces yeast strains were evaluated in industrially relevant media. High-throughput robotic platforms, the ROTOR HDA and PIXL, were used to pin colonies on solidified synthetic apple juice (SAJ), malt extract (ME), apple juice (AJ) and wort (WO) media. During incubation at 20 and 25 °C for up to 240 hours, the plates were imaged, and analysed via PhenoBooth and the ggplot2 R package. The results were corroborated via liquid growth. Pichia scullata Y-7663 and Hanseniaspora vineae Y-17530 showed the highest growth values in ME, WO and AJ; with final colony diameters double that of the Saccharomyces cerevisiae ESM356-1 control. However, the former two strains displayed defective growth in SAJ. Kluyveromyces lactis Y-1140 exhibited high growth performance, especially in ME and WO. Interestingly, the ale strain S. cerevisiae Y-11875, showed significantly higher growth in SAJ and AJ medium than in ME and WO. Better adaptation to growth in wort conditions was expected. This trial shows that the fitness of yeast strains can be assessed in solid media representing industrial conditions. The method can be up-scaled to a wider format, opening the possibility of screening a thousand-fold libraries of industrially relevant and/or novel yeast isolates in a fast, cost-effective manner.