Development of a CRISPR/Cas9-Based Tool for Gene Deletion in Issatchenkia orientalis

ABSTRACT The nonconventional yeast Issatchenkia orientalis has emerged as a potential platform microorganism for production of organic acids due to its ability to grow robustly under highly acidic conditions. However, lack of efficient genetic tools remains a major bottleneck in metabolic engineering of this organism. Here we report that the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1′-tRNA promoter for single guide RNA (sgRNA) expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. This genome editing tool can be used for rapid strain development and metabolic engineering of this organism for production of biofuels and chemicals. IMPORTANCE Microbial production of fuels and chemicals from renewable and readily available biomass is a sustainable and economically attractive alternative to petroleum-based production. Because of its unusual tolerance to highly acidic conditions, I. orientalis is a promising potential candidate for the manufacture of valued organic acids. Nevertheless, reliable and efficient genetic engineering tools in I. orientalis are limited. The results outlined in this paper describe a stable episomal ARS-containing plasmid and the first CRISPR/Cas9-based system for gene disruptions in I. orientalis, paving the way for applying genome engineering and metabolic engineering strategies and tools in this microorganism for production of fuels and chemicals.

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Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

mSphere ◽

10.1128/msphere.00130-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 68

Author(s):

Kyunghun Min ◽

Yuichi Ichikawa ◽

Carol A. Woolford ◽

Aaron P. Mitchell

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Genetic Analysis ◽

Genome Editing ◽

Drug Targets ◽

Gene Deletion ◽

Genetic Manipulation ◽

Content Type ◽

Biological Features ◽

Link Type

ABSTRACT The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

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Efficient Genome Engineering of a Virulent Klebsiella Bacteriophage Using CRISPR-Cas9

Journal of Virology ◽

10.1128/jvi.00534-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 11

Author(s):

Juntao Shen ◽

Jinjie Zhou ◽

Guo-Qiang Chen ◽

Zhi-Long Xiu

Keyword(s):

Genome Editing ◽

Point Mutation ◽

Gene Deletion ◽

Phage Therapy ◽

Phage Genome ◽

Cost Effective ◽

Bacteriophage Therapy ◽

Time Saving ◽

Content Type ◽

Frameshift Deletion

ABSTRACT Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens and usually exhibits multiple-drug resistance. Phage therapy, a potential therapeutic to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well characterized because of the lack of efficient genome-editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage, phiKpS2. We first evaluated the distribution of sgRNA activity in phages and proved that it is largely inconsistent with the predicted activity from current models trained on eukaryotic cell data sets. A simple CRISPR-based phage genome-editing procedure was developed based on the discovery that homologous arms as short as 30 to 60 bp were sufficient to introduce point mutation, gene deletion, and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the abovementioned strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy. IMPORTANCE In the present study, we have addressed efficient, time-saving, and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proven to be enough to introduce point mutation, small frameshift deletion, gene deletion, and swap into phages, and weak sgRNAs were proven useful for precise phage genome editing but failed to select random recombinants, all of which makes the CRISPR-based phage genome-editing method easier to use.

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Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides

mSphere ◽

10.1128/msphere.00099-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 17

Author(s):

Peter B. Otoupal ◽

Masakazu Ito ◽

Adam P. Arkin ◽

Jon K. Magnuson ◽

John M. Gladden ◽

...

Keyword(s):

Genome Editing ◽

Gene Disruption ◽

Genome Engineering ◽

Carbon Sources ◽

Rhodosporidium Toruloides ◽

Optimized Design ◽

Content Type ◽

Wide Range ◽

Fuels And Chemicals ◽

Microbial Biofuel

ABSTRACT Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides’ engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides. Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism. IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast’s production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

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Forced Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene Deletion/Integration in the Industrial Filamentous Fungus Aspergillus oryzae

Applied and Environmental Microbiology ◽

10.1128/aem.01896-18 ◽

2018 ◽

Vol 85 (3) ◽

Cited By ~ 11

Author(s):

Takuya Katayama ◽

Hidetoshi Nakamura ◽

Yue Zhang ◽

Arnaud Pascal ◽

Wataru Fujii ◽

...

Keyword(s):

Secondary Metabolites ◽

Genetic Engineering ◽

Genome Editing ◽

Aspergillus Oryzae ◽

Filamentous Fungus ◽

Gene Deletion ◽

Multiple Gene ◽

Content Type ◽

Industrial Strains ◽

Marker Free

ABSTRACT Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established. We have previously developed a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9-based mutagenesis technique for the industrial filamentous fungus Aspergillus oryzae, enabling mutation efficiency of 10 to 20%. Here, we improved the CRISPR/Cas9 approach by including an AMA1-based autonomously replicating plasmid harboring the drug resistance marker ptrA. By using the improved mutagenesis technique, we successfully modified A. oryzae wild and industrial strains, with a mutation efficiency of 50 to 100%. Conditional expression of the Aoace2 gene from the AMA1-based plasmid severely inhibited fungal growth. This enabled forced recycling of the plasmid, allowing repeated genome editing. Further, double mutant strains were successfully obtained with high efficiency by expressing two guide RNA molecules from the genome-editing plasmid. Cotransformation of fungal cells with the genome-editing plasmid together with a circular donor DNA enabled marker-free multiplex gene deletion/integration in A. oryzae. The presented repeatable marker-free genetic engineering approach for mutagenesis and gene deletion/integration will allow for efficient modification of multiple genes in industrial fungal strains, increasing their applicability. IMPORTANCE Multiple gene modifications of specific fungal strains are required for achieving industrial-scale production of enzymes and secondary metabolites. In the present study, we developed an efficient multiple genetic engineering technique for the filamentous fungus Aspergillus oryzae. The approach is based on a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and recycling of an AMA1-based autonomous replicating plasmid. Because the plasmid harbors a drug resistance marker (ptrA), the approach does not require the construction of auxotrophic industrial strains prior to genome editing and allows for forced recycling of the gene-editing plasmid. The established plasmid-recycling technique involves an Aoace2-conditional expression cassette, whose induction severely impairs fungal growth. We used the developed genetic engineering techniques for highly efficient marker-free multiple gene deletion/integration in A. oryzae. The genome-editing approaches established in the present study, which enable unlimited repeatable genetic engineering, will facilitate multiple gene modification of industrially important fungal strains.

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Development of a CRISPR-Cas9 System for Efficient Genome Editing of Candida lusitaniae

mSphere ◽

10.1128/msphere.00217-17 ◽

2017 ◽

Vol 2 (3) ◽

Cited By ~ 14

Author(s):

Emily L. Norton ◽

Racquel K. Sherwood ◽

Richard J. Bennett

Keyword(s):

Genome Editing ◽

Gene Targeting ◽

Gene Deletion ◽

Human Pathogen ◽

Mechanistic Studies ◽

Content Type ◽

Dna Dsbs ◽

Candida Lusitaniae ◽

Species Specific ◽

Genome Manipulation

ABSTRACT The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C. lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C. lusitaniae biology. Candida lusitaniae is a member of the Candida clade that includes a diverse group of fungal species relevant to both human health and biotechnology. This species exhibits a full sexual cycle to undergo interconversion between haploid and diploid forms. C. lusitaniae is also an emerging opportunistic pathogen that can cause serious bloodstream infections in the clinic and yet has often proven to be refractory to facile genetic manipulations. In this work, we develop a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (Cas9) system to enable genome editing of C. lusitaniae. We demonstrate that expression of CRISPR-Cas9 components under species-specific promoters is necessary for efficient gene targeting and can be successfully applied to multiple genes in both haploid and diploid isolates. Gene deletion efficiencies with CRISPR-Cas9 were further enhanced in C. lusitaniae strains lacking the established nonhomologous end joining (NHEJ) factors Ku70 and DNA ligase 4. These results indicate that NHEJ plays an important role in directing the repair of DNA double-strand breaks (DSBs) in C. lusitaniae and that removal of this pathway increases integration of gene deletion templates by homologous recombination. The described approaches significantly enhance the ability to perform genetic studies in, and promote understanding of, this emerging human pathogen and model sexual species. IMPORTANCE The ability to perform efficient genome editing is a key development for detailed mechanistic studies of a species. Candida lusitaniae is an important member of the Candida clade and is relevant both as an emerging human pathogen and as a model for understanding mechanisms of sexual reproduction. We highlight the development of a CRISPR-Cas9 system for efficient genome manipulation in C. lusitaniae and demonstrate the importance of species-specific promoters for expression of CRISPR components. We also demonstrate that the NHEJ pathway contributes to non-template-mediated repair of DNA DSBs and that removal of this pathway enhances efficiencies of gene targeting by CRISPR-Cas9. These results therefore establish important genetic tools for further exploration of C. lusitaniae biology.

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Transcriptional Regulation of Plant Biomass Degradation and Carbohydrate Utilization Genes in the Extreme Thermophile Caldicellulosiruptor bescii

mSystems ◽

10.1128/msystems.01345-20 ◽

2021 ◽

Cited By ~ 1

Author(s):

Dmitry A. Rodionov ◽

Irina A. Rodionova ◽

Vladimir A. Rodionov ◽

Aleksandr A. Arzamasov ◽

Ke Zhang ◽

...

Keyword(s):

Metabolic Engineering ◽

Plant Biomass ◽

Biomass Degradation ◽

Extreme Thermophile ◽

Comprehensive Understanding ◽

Carbohydrate Utilization ◽

Content Type ◽

Metabolic Characteristics ◽

Caldicellulosiruptor Bescii ◽

Fuels And Chemicals

To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium.

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Pgas, a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger

Applied and Environmental Microbiology ◽

10.1128/aem.03222-16 ◽

2017 ◽

Vol 83 (6) ◽

Cited By ~ 22

Author(s):

Xian Yin ◽

Hyun-Dong Shin ◽

Jianghua Li ◽

Guocheng Du ◽

Long Liu ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Engineering ◽

Cell Growth ◽

Aspergillus Niger ◽

Organic Acids ◽

Dynamic Control ◽

Low Ph ◽

Transcriptional Analysis ◽

Control Of Gene Expression ◽

Content Type

ABSTRACT The dynamic control of gene expression is important for adjusting fluxes in order to obtain desired products and achieve appropriate cell growth, particularly when the synthesis of a desired product drains metabolites required for cell growth. For dynamic gene expression, a promoter responsive to a particular environmental stressor is vital. Here, we report a low-pH-inducible promoter, Pgas, which promotes minimal gene expression at pH values above 5.0 but functions efficiently at low pHs, such as pH 2.0. First, we performed a transcriptional analysis of Aspergillus niger, an excellent platform for the production of organic acids, and we found that the promoter Pgas may act efficiently at low pH. Then, a gene for synthetic green fluorescent protein (sGFP) was successfully expressed by Pgas at pH 2.0, verifying the results of the transcriptional analysis. Next, Pgas was used to express the cis-aconitate decarboxylase (cad) gene of Aspergillus terreus in A. niger, allowing the production of itaconic acid at a titer of 4.92 g/liter. Finally, we found that Pgas strength was independent of acid type and acid ion concentration, showing dependence on pH only. IMPORTANCE The promoter Pgas can be used for the dynamic control of gene expression in A. niger for metabolic engineering to produce organic acids. This promoter may also be a candidate tool for genetic engineering.

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Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

Journal of Fungi ◽

10.3390/jof7020149 ◽

2021 ◽

Vol 7 (2) ◽

pp. 149

Author(s):

Sarah-Maria Wege ◽

Katharina Gejer ◽

Fabienne Becker ◽

Michael Bölker ◽

Johannes Freitag ◽

...

Keyword(s):

Genome Editing ◽

Cell Biology ◽

Gene Deletion ◽

Fluorescent Protein ◽

Point Mutations ◽

Model Organism ◽

Ustilago Maydis ◽

Genomic Locus ◽

Targeted Gene Deletion ◽

Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

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Normal peripheral blood neutrophil numbers accompanying ELANE whole gene deletion mutation

Blood Advances ◽

10.1182/bloodadvances.2019000498 ◽

2019 ◽

Vol 3 (16) ◽

pp. 2470-2473 ◽

Cited By ~ 2

Author(s):

Marshall S. Horwitz ◽

Mercy Y. Laurino ◽

Siobán B. Keel

Keyword(s):

Genome Editing ◽

Peripheral Blood ◽

Gene Deletion ◽

Deletion Mutation ◽

Blood Neutrophil ◽

Normal Peripheral Blood ◽

Peripheral Blood Neutrophil ◽

Key Points ◽

Patient Reported

Key Points The patient reported here, along with collective observations in the literature, suggest that ELANE deletion does not cause neutropenia. Potential therapeutic genome editing involving knockout of the mutant ELANE allele is therefore not expected to produce neutropenia.

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An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽

10.1128/mspheredirect.00149-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 33

Author(s):

Namkha Nguyen ◽

Morgan M. F. Quail ◽

Aaron D. Hernday

Keyword(s):

Candida Albicans ◽

Genome Editing ◽

Fungal Pathogen ◽

Gene Knockout ◽

Strain Engineering ◽

Molecular Genetic Analysis ◽

Content Type ◽

Highly Efficient ◽

Discovery Research ◽

Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

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