ADE2 Gene Research Articles

Development of ptxD/Phi as a new dominant selection system for genetic manipulation in Cryptococcus neoformans.

Cryptococcus neoformans is a globally distributed pathogenic fungus posing a significant threat to immunocompromised individuals, particularly those with HIV/AIDS. Effective genetic manipulation tools are essential for understanding its biology and developing new therapies. However, current genetic tools, including the variation of versatile selectable markers, are limited. This study develops and validates the phosphite dehydrogenase gene (ptxD)/phosphite (Phi) selection system as a non-antibiotic selectable marker for genetic manipulation in C. neoformans. A codon-optimized ptxD gene from Pseudomonas stutzeri was cloned under the TEF promoter. Using the transient CRISPR-Cas9 coupled with electroporation system, we integrated the ptxD gene into the C. neoformans genome and assessed the impact of ptxD integration on cell growth and virulence factors. The ptxD/Phi system effectively selected transformed cells on Phi-containing media. Growth assays showed that ptxD integration did not adversely affect cell growth or key virulence factors, including pleomorphism, capsule size, and melanin production. Additionally, we successfully disrupted the ADE2 gene using this system, confirming its applicability for gene deletion. Taken together, the ptxD/Phi system provides a robust and versatile tool for genetic manipulation in C. neoformans, facilitating further research into its biology and pathogenicity.IMPORTANCECryptococcus neoformans is a type of fungus that can cause serious illnesses in people who have weakened immune systems, like those with HIV/AIDS. To better study this fungus and find new treatments, scientists need tools to change its genes in precise ways. However, the current tools available for this are somewhat limited. This research introduces a new tool called the phosphite dehydrogenase gene/phosphite system, which does not rely on antibiotics to work. It uses a gene from a different bacterium that helps select and grow only the fungus cells that have successfully incorporated new genetic information. This is particularly useful because it does not interfere with the normal growth of the fungus or the features that make it harmful (like its ability to change shape or produce protective coatings). By making it easier and more effective to manipulate the genetics of C. neoformans, this tool opens up new possibilities for understanding how this fungus operates and for developing therapies to combat its infections. This is crucial for improving the treatment of infections in vulnerable populations.

Design and practice of educational experiments on genetic epistasis.

Genetic epistasis is a fundamental concept in genetics that describes how interactions between genes determine phenotypic traits. To enhance students' understanding and practical application of genetic epistasis, this experiment is designed and conducted using gene mutations in the adenine biosynthesis pathway of Saccharomyces cerevisiae (baker's yeast). S. cerevisiae is a classic model organism for genetic teaching experiments. In its adenine biosynthesis pathway, a mutation in the ADE2 gene leads to the accumulation of the intermediate 5'-phosphoribosylaminoimidazole (AIR), causing the cells to appear red. However, if a gene upstream of ADE2 in the adenine biosynthesis pathway (such as ADE8) is defective, the red phenotype of yeast will disappear. Conversely, a defect in a gene downstream of ADE2 (such as ADE1) does not alter the red phenotype. Therefore, ADE8 is epistatic to ADE2. In this experiment, the CRISPR-Cas9 genome editing technology is employed, allowing students to perform single knockout of ade2Δ, as well as double knockouts of ade2Δade8Δ and ade2Δade1Δ in S. cerevisiae. By observing the phenotypic changes in yeast mutants from white to red and back to white, students gain a profound understanding of the basic genetic theory of how genes determine phenotypes and the concept of epistasis in gene interactions. This experiment also enables students to master fundamental yeast genetic techniques, significantly enhancing their ability to design and conduct experiments in real research environments. This is of great significance for their future research work and academic development.

Creating Meiotic Recombination-Regulating DNA Sites by SpEDIT in Fission Yeast Reveals Inefficiencies, Target-Site Duplications, and Ectopic Insertions.

Recombination hotspot-activating DNA sites (e.g., M26, CCAAT, Oligo-C) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the ade6 gene of Schizosaccharomyces pombe. We used a fission yeast-optimized CRISPR-Cas9 system (SpEDIT) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at ade6. PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that SpEDIT can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus.

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.

Genome editing toolboxes are essential for the exploration and exploitation of non-conventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The non-conventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1,000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses non-homologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.

Expanding the genome editing toolbox of Saccharomyces cerevisiae with the endonuclease ErCas12a.

ErCas12a is a class 2 type V CRISPR-Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces cerevisiae. The optimal design parameters for ErCas12a editing in S. cerevisiae were defined as a 21-nt spacer flanked by 19nt direct repeats expressed from either RNApolII or III promoters, achieving near 100% editing efficiencies in commonly targeted genomic locations. To be able to transfer the ErCas12a genome editing tool to different strain lineages, a transportable platform plasmid was constructed and evaluated for its genome editing efficiency. Using an identical crRNA expression design, the transportable ErCas12a genome editing tool showed lower efficiency when targeting the ADE2 gene. In contrast to genomic Ercas12a expression, episomal expression of Ercas12a decreases maximum specific growth rate on glucose, indicating ErCas12a toxicity at high expression levels. Moreover, ErCas12a processed a multispacer crRNA array using the RNA self-processing capability, which allowed for simultaneous editing of multiple chromosomal locations. ErCas12a is established as a valuable addition to the genetic toolbox for S. cerevisiae.

PCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

CRISPR-Cas9 technology is widely used for precise and specific editing of Saccharomyces cerevisiae genome to obtain marker-free engineered hosts. Targeted double-strand breaks are controlled by a guide RNA (gRNA), a chimeric RNA containing a structural segment for Cas9 binding and a 20-mer guide sequence that hybridises to the genomic DNA target. Introducing the 20-mer guide sequence into gRNA expression vectors often requires complex, time-consuming, and/or expensive cloning procedures. We present a new plasmid for CRISPR-Cas9 genome editing in S. cerevisiae, pCEC-red. This tool allows to (i) transform yeast with both Cas9 and gRNA expression cassettes in a single plasmid and (ii) insert the 20-mer sequence in the plasmid with high efficiency, thanks to Golden Gate Assembly and (iii) a red chromoprotein-based screening to speed up the selection of correct plasmids. We tested genome-editing efficiency of pCEC-red by targeting the ADE2 gene. We chose three different 20-mer targets and designed two types of repair fragments to test pCEC-red for precision editing and for large DNA region replacement procedures. We obtained high efficiencies (∼90%) for both engineering procedures, suggesting that the pCEC system can be used for fast and reliable marker-free genome editing.

A simple and accessible CRISPR genome editing laboratory exercise using yeast.

CRISPR is a revolutionary tool to engineer the genome. Herein, we describe a laboratory exercise designed to introduce students to CRISPR by editing the genome of yeast to disrupt the ADE2 gene, which results in yeast that form red colored colonies. The experiment was constructed to be performed in a single laboratory session and to be accessible to students and instructors without experience working with yeast.

Genetically modified yeasts in studies of human amyloidosis

Amyloid protein aggregation is a key factor in the development of a variety of serious diseases in humans, commonly named as amyloidoses (Alzheimers and Parkinsons diseases, type II diabetes, etc.), and a determinant of protein-based inheritance in lower eukaryotes. In yeast, translation termination factor Sup35 is one of the most extensively studied amyloidogenic proteins. Aggregation of Sup35 (induction of [PSI+] prion) decreases its functional activity and leads to the suppression of nonsense-mutation as stop-codons become recognized as meaningful more frequently. This phenomenon is the basis of phenotypic detection of Sup35 aggregation in yeast strains possessing nonsense mutation ade1-14 in ADE1 gene.&#x0D; Yeast is convenient model for genetic, biochemical and molecular biology studies. Yeast genome can be easily edited and plasmids can be used for induction of gene expression. Yeast is suitable for analysis of mammalian genes and proteins and thus can be applied for the analysis of amyloidogenic properties of proteins associated with human diseases. Phenotyping detection of [PSI+] prion can be modified for the analysis of amyloid aggregation of mammalian proteins in yeast.&#x0D; We use genetically modified yeasts Saccharomyces cerevisiae adopted for amyloid biology research. The mutations leading to auxotrophy toward certain amino acids (leucine, lysine, tryptophane, histidine) and nucleobases (adenine, uracil) were implemented into yeast genome allowing phenotyping detection of [PSI+] and the usage of plasmids for the investigation of mammalian protein in yeast.&#x0D; Application of yeast-based experimental system for studies of different aspects of human amyloidoses is discussed.&#x0D; This study was supported by Saint Petersburg State University (project 93025998).

A Test System for Assessment of the Activity of Mutant Cas9 Variants in Saccharomyces cerevisiae

The key component of the revolutionary Streptococcus pyogenes CRISPR/Cas genome editing technology is the multidomain protein Cas9. However, the specificity of wild type Cas9 is not sufficiently high for editing large genomes of higher eukaryotes, which limits the realization of the potential of genomic editing both in fundamental investigations and in the therapy of genetic diseases. The main way to obtain more specific variants of Cas9 is through mutagenesis followed by characterization of mutant proteins in in vitro or in vivo test systems. The in vitro and some in vivo test systems described in the literature are often labor-intensive and have scaling limitations, which makes it challenging to screen SpCas9 mutant variant libraries. In order to develop a simple method for high-throughput screening of Cas9 mutants in vivo, we characterized three test systems using CRISPR/Cas9-mediated inactivation of the reporter genes, tsPurple, ADE2, and URA3, in the Saccharomyces cerevisiae yeast as a model subject. We measured the activities of high-precision forms of Cas9, evoCas9, and HiFiCas9, and compared them with the wild-type form. ADE2 gene inactivation was found to be the most valid method for the evaluation of Cas9 activity. In the test-system developed, the sensitivity to chromatin structure was demonstrated for the high-fidelity variant of Cas9, HiFiCas9. The proposed test-system can be used for the development of new generation genome editors.

Highly selective and potent p16-CDK-RB-E2F targeted oncolytic virus therapies.

e14543 Background: Growth factor and p16-CDK-RB-E2F pathway components are mutated in almost all cancers, resulting in oncogenic E2F transcriptional activity that drives uncontrolled proliferation. Chemotherapies indiscriminately kill all proliferating cells, resulting in limiting toxicities. CDK inhibitors are more targeted but cytostatic as opposed to tumoricidal. E2F was discovered as a factor that transcribes adenovirus (Ad) E2 and cellular genes to drive viral replication. Ad E1A binds to RB, which activates E2F. On this basis, viruses with E1A-RB binding mutations are being evaluated in the clinic as oncolytic viruses (OVs) that selectively replicate in and kill RB mutant tumor cells. However, E1A-RB mutations alone are not sufficient to prevent viral replication in normal cells. The addition of ‘tumor specific promoters’ improves tumor selectivity but drastically impacts replication and lytic activity. Therefore, a critical unmet need is the engineering of E2F addicted OVs that replicate like wildtype viruses in tumor cells, but leave normal cells unharmed. Methods: We have developed a proprietary platform that enables Ad therapeutics to be assembled from libraries of functional genomic parts that confer desirable properties. Here we describe E1 and E4 genomic modules that confer E2F dependent tumor selective replication. We assembled a panel of ̃50 viruses with combinations of mutations that ablate i) E1A interactions with RB/p107/p130 and/or EP300 ii) E4-ORF1 activation of PI3-Kinase and/or MYC iii) E4-ORF6/7 induced E2F1/DP1 dimerization. These viruses were screened in panels of primary, tumor, and CRISPR-engineered cells. Results: These studies reveal that E4-ORF6/7 functions independently of E1A to activate E2F transcriptional targets, S phase entry and viral replication. We show that viruses with both E1A and E4-ORF6/7 mutations (AdSyn-181) have the ideal tumor selectivity/efficacy profiles and outperform existing OVs. We also show that the deletion of p16 in primary cells rescues AdSyn-181 replication by ̃15 fold (̃20% wildtype virus), demonstrating the E2F dependence and tumor selectivity mechanism. Furthermore, we show that in tumor cells, which have multiple p16-CDK-RB pathway mutations, AdSyn-181 replication is rescued to 100% wildtype virus levels. ICVB-1042, which incorporates these selectivity mutations – together with other modules that enhance lytic cell death and spread, tumor tropisms and IV delivery – is in IND-enabling studies. Conclusions: Leveraging extensive research into the overlapping logic of viral and cancer pathways, we have engineered OVs, including ICVB-1042, that distinguish between tumor and normal cell proliferation. Furthermore, unlike therapies that target a single component, ICVB-1042 targets tumors with different p16-CDK-RB mutations, which all converge in activating oncogenic E2F transcription, driving exponential and tumoricidal viral replication.

Targeted Forward Genetics: Population-Scale Analyses of Allele Replacements Spanning Thousands of Base Pairs in Fission Yeast.

Precise allele replacement (genome editing), without unwanted changes to the genome, provides a powerful tool to define the functions of DNA elements and encoded factors in their normal biological context. While CRISPR is now used extensively for gene targeting, its utility for precise allele replacement at population scale is limited because: (A) there is a strict requirement for a correctly positioned PAM motif to introduce recombinogenic dsDNA breaks (DSBs); (B) efficient replacements only occur very close to the DSBs; and (C) indels and off-target changes are frequently generated. Here we show, using a saturated mutation library with about 15,000 alleles of the ade6 gene of Schizosaccharomyces pombe, that pop-in, pop-out allele replacement circumvents these problems. Two rounds of selection ensure that clones arise by homologous recombination with the target locus. Moreover, the exceptionally high efficiency allows one to carry out the process in bulk, then screen individual clones for phenotypes and genotypes. Alleles were introduced successfully throughout the region targeted, up to 1,956 base pairs from the DSB. About 11% of mutant alleles were hypomorphic, demonstrating utility for analyses of essential genes and genetic elements. This process of “targeted forward genetics” can be used to analyze comprehensively, across thousands of base pairs within a specific target region, a variety of allelic changes, such as scanning amino acid substitutions, deletions, and epitope tags. The overall approach and optimized workflow are extensible to other organisms that support gene targeting.

Nuclear export signal (NES) of transposases affects the transposition activity of mariner-like elements Ppmar1 and Ppmar2 of moso bamboo

Ppmar1 and Ppmar2 are two active mariner-like elements (MLEs) cloned from moso bamboo (Phyllostachys edulis (Carrière) J. Houz) genome possessing transposases that harbour nuclear export signal (NES) domain, but not any nuclear localization signal (NLS) domain. To understand the functions of NES in transposon activity, we have conducted two experiments, fluorescence and excision frequency assays in the yeast system. For this, by site-directed mutagenesis, three NES mutants were developed from each of the MLE. In the fluorescence assay, the mutants, NES-1, 2 and 3 along with the wild types (NES-0) were fused with fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were co-transformed into yeast system. To differentiate protein localisation under the NES influence, ECFP alone was fused to wild and mutant NES domains either on N- or C-terminal and not to EYFP. Fluorescence assay revealed that blue fluorescence of ECFP was more intense than the red fluorescence of the EYFP in the yeast cell matrix. Further, ECFP had a wider localisation in the cellular matrix, but EYFP was largely located in the nucleus. The NES-1 domain was related to the comparatively high spread of ECFP, while NES-2 and NES-3 indicated a low spread, implying that NES activity on nuclear export increased when the NES is made leucine-rich, while the signalling activity was reduced when the leucine content was lowered in the NES domain. In the transposon excision assay, the mutant and wild type NES of both the Ppmar elements were integrated into an Ade2 vector, and within the Ade2 gene. Co-transformation of the vector together with non-autonomous Ppmar transposons and NES-lacking transposases was used to assess the differential excision frequencies of the mutants NES domains. In both the MLEs, NES-1 had the highest excision suppression, which was less than half of the excision frequency of the wild type. NES-2 and NES-3 elements showed, up to three times increase in transposon excision than the wild types. The results suggested that NES is an important regulator of nuclear export of transposase in Ppmar elements and the mutation of the NES domains can either increase or decrease the export signalling. We speculate that in moso bamboo, NESs regulates the transposition activity of MLEs to maintain the genome integrity.

How to make red yeast: An undergraduate laboratory using CRISPR/Cas9 technology to target ADE2 in budding yeast

The ability of CRISPR/Cas9 to edit genomic sequence in a multitude of organisms has revolutionized modern science and is an ideal topic to engage undergraduate students. The ease and speed of this technology combined with the short generation time, well‐characterized genome, established protocols, and relatively limited financial demands of Saccharomyces cerevisiae (bakers yeast) make for a practical undergraduate laboratory. Students, working in pairs were assigned the task of replacing the chromosomal copy of ADE2 with HIS3. Over the course of several weeks students cloned the ADE2 gRNA into an expression vector, PCR amplified a HIS3 construct for homologous recombination, and transformed these components into yeast. ADE2 encodes for Phosphoribosylaminoimidazole carboxylase, an enzyme that catalyzes a reaction in the de novo synthesis of purine nucleotides. Disruption of this gene results in the accumulation of red pigment in the yeast cells. Students were thus able to screen for disruption of the ADE2 gene and subsequent replacement with HIS3 with a specific growth phenotype: red yeast capable of growing on media lacking histidine. The methods used in this lab included: basic cell culture, primer design, PCR, plasmid isolation, transformation, restriction digestion, ligation and DNA sequencing. To reinforce scientific writing and analysis skills the culmination of this project was a manuscript describing their results and a final class lab meeting where each group presented their data and conclusions. Informal assessments indicated that participating in this lab increased student's interest in attending graduate schools and pursing careers in biomedical research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acetamidase as a dominant recyclable marker for Komagataella phaffii strain engineering.

We have investigated the use of the gene coding for acetamidase (amdS) as a recyclable dominant marker for the methylotrophic yeast Komagataella phaffii in order to broaden its genetic toolbox. First, the endogenous constitutive AMD2 gene (a putative acetamidase) was deleted generating strain LA1. A cassette (amdSloxP) was constructed bearing a codon-optimized version of the Aspergillus nidulans amdS gene flanked by loxP sites for marker excision with Cre recombinase. This cassette was successfully tested as a dominant selection marker for transformation of the LA1 strain after selection on plates containing acetamide as a sole nitrogen source. Finally, amdSloxP was used to sequentially disrupt the K. phaffii ADE2 and URA5 genes. After each disruption event, a Cre-mediated marker recycling step was performed by plating cells on medium containing fluoroacetamide. In conclusion, amdS proved to be a suitable tool for K. phaffii transformation and marker recycling thus providing a new antibiotic-free system for genetic manipulation of this yeast.

Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade− mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.

Distinct mechanisms of phenotypic effects of inactivation and prionization of Swi1 protein in Saccharomyces cerevisiae

Prions are proteins that under the same conditions can exist in two or more conformations, and at least one of the conformations has infectious properties. The prionization of a protein is typically accompanied by its functional inactivation due to sequestration of monomers by the prion aggregates. The most of prions has been identified in the yeast Saccharomyces cerevisiae. One of them is [SWI+], a prion isoform of the Swi1 protein, which is a component of the evolutionarily conserved chromatin remodeling complex SWI/SNF. Earlier, it was shown that the prionization of [SWI+] induces a nonsense suppression, which leads to weak growth of the [SWI+] strains containing mutant variants of the SUP35 gene and the nonsense allele ade1-14UGA on selective medium without adenine. This effect occurs because of [SWI+] induction that causes a decrease in the amount of the SUP45 mRNA. Strains carrying the SWI1 deletion exhibit significantly higher suppression of the ade1-14UGA nonsense mutation than the [SWI+] strains. In the present study, we identified genes whose expression is altered in the background of the SWI1 deletion using RNA sequencing. We found that the ade1-14UGA suppression in the swi1Δ strains is caused by an increase in the expression of this mutant allele of the ADE1 gene. At the same time, the SUP45 expression level in the swi1Δ strains does not significantly differ from the expression level of this gene in the [swi-] strains. Thus, we have shown that the phenotypic effects of Swi1 prionization and deletion are mediated by different molecular mechanisms. Based on these data, we have concluded that the prionization of proteins is not only unequal to their inactivation, but also can lead to the acquisition of novel phenotypic effects and functions.

A new type of gene-disruption cassette with a rescue gene for Pichia pastoris.

Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter-driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant-loxP sequences in the cassette is excised, leaving only the remaining mutant-loxP sequence in the genome, and consequently a target gene-disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color-change of the colonies. Then, the system was applied for knocking-out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene-targeting methods. All three gene-disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant-loxP system worked well to successfully isolate their knock-out mutants. This study identified a new gene-disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end-joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201-1208, 2017.

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats.

Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes.

Targeted Genome Editing via CRISPR in the Pathogen Cryptococcus neoformans.

Low rates of homologous integration have hindered molecular genetic studies in Cryptococcus neoformans over the past 20 years, and new tools that facilitate genome manipulation in this important pathogen are greatly needed. To this end, we have investigated the use of a Class 2 CRISPR system in C. neoformans (formerly C. neoformans var. grubii). We first expressed a derivative of the Streptococcus pyogenes Cas9 nuclease in C. neoformans, and showed that it has no effect on growth, production of virulence factors in vitro, or virulence in a murine inhalation model. In proof of principle experiments, we tested the CAS9 construct in combination with multiple self-cleaving guide RNAs targeting the well-characterized phosphoribosylaminoamidazole carboxylase-encoding ADE2 gene. Utilizing combinations of transient and stable expression of our constructs, we revealed that functionality of our CRISPR constructs in C. neoformans is dependent upon the CAS9 construct being stably integrated into the genome, whilst transient expression of the guide RNA is sufficient to enhance rates of homologous recombination in the CAS9 genetic background. Given that the presence of the CRISPR nuclease does not influence virulence in a murine inhalation model, we have successfully demonstrated that this system is compatible with studies of C. neoformans pathogenesis and represents a powerful tool that can be exploited by researchers in the field.

Unique properties of multiple tandem copies of the M26 recombination hotspot in mitosis and meiosis in Schizosaccharomyces pombe

The M26 hotspot of the fission yeast Schizosaccharomyces pombe is one of the best-characterized eukaryotic hotspots of recombination. The hotspot requires a seven bp sequence, ATGACGT, that serves as a binding site for the Atf1-Pcr1 transcription factor, which is also required for activity. The M26 hotspot is active in meiosis but not mitosis and is active in some but not all chromosomal contexts and not on a plasmid. A longer palindromic version of M26, ATGACGTCAT, shows significantly greater activity than the seven bp sequence. Here, we tested whether the properties of the seven bp sequence were also true of the longer sequence by placing one, two, or three copies of the sequence into the ade6 gene, where M26 was originally discovered. These constructs were tested for activity when located on a plasmid or on a chromosome in mitosis and meiosis. We found that two copies of the 10bp M26 motif on a chromosome were significantly more active for meiotic recombination than one, but no further increase was observed with three copies. However, three copies of M26 on a chromosome created an Atf1-dependent mitotic recombination hotspot. When located on a plasmid, M26 also appears to behave as a mitotic recombination hotspot; however, this behavior most likely results from Atf1-dependent inter-allelic complementation between the plasmid and chromosomal ade6 alleles.