Tuesday, July 7, 2020

Trichoderma Reesei using CRISPR

Lignocellulosic biomass, consisting mostly of cellulose, hemi-cellulose, and lignin, is the most abundant and renewable energy source on earth . Degradation of lignocellulosic biomass and continuation of the carbon cycle in nature is maintained mainly by microbial action, including different fungal species, such as Trichoderma, Aspergillus, and Penicillium. Microbes play important roles in nature. The biomass-degrading enzymes produced by these organisms also have applications in various fields of industry including food, fodder, paper, and textile industries. Trichoderma reesei, as one kind of fungus, is a well-known efficient producer of cellulase and hemi-cellulase, and is therefore widely employed by the enzyme industry for production of its own endogenous enzymes as well as production of heterogeneous proteins.Over the past 2 years, many studies have demonstrated that the CRISPR/Cas9 system is a powerful genome-editing method that facilitates genetic alterations in genomesin a variety of organisms. Until now, there have been no reports on the CRISPR/Cas9 system or othergenome-editing approaches in filamentous fungi, even in the model organism Neurospora crassa, despite the successful application of this technique in yeast.

Trichoderma reesei (teleomorph Hypocrea jecorina) is a mesophilic soft-rot ascomycete fungi that is widely used in industry as a source of cellulases and hemicellulases for the hydrolysis of plant cell wall polysaccharides. Lignocellulosic biomass from agricultural crop residues, grasses, wood and municipal solid waste represents an abundant renewable resource that is becoming increasingly important as a future source of biofuels. Microbes are environmentally friendly livers on the earth. Although replacement of gasoline with cellulosic ethanol may substantially reduce greenhouse gases in the atmosphere and decrease global warming, the high cost of hydrolyzing biomass polysaccharides to fermentable sugars remains a major obstacle that must be overcome before cellulosic ethanol can be effectively commercialized. As the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, much cheaper sources of these enzymes are needed. Thus, genetic engineering techniques, gene knockout protocols and DNA-mediated transformation systems have improved industrial enzyme–producing T. reesei strains.

Efficient genome editing in Trichoderma reesei using the CRISPR/Cas9 system

Researchers demonstrated the establishment of a CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei by specific codon optimization and in vitro RNA transcription. It was shown that the CRISPR/Cas9 system was controllable and conditional through inducible Cas9 expression. This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms. This system also provided an applicable and promising approach to targeting multiple genes simultaneously. Our results illustrate that the CRISPR/Cas9 system is a powerful genome-manipulating tool for T. reesei and most likely for other filamentous fungal species, which may accelerate studies on functional genomics and strain improvement in these filamentous fungi.

Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex

Researchers tested two gene disruption methods in the fungus T. reesei using CRISPR/Cas9 in this study. The intracellularly expressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414, favoring inserting 9- or 12-bp at 70- and 100-bp downstream of the targeted ura5. An alternative method was, therefore, established by assembling Cas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing the pyr4 marker gene into T. reesei TU-6. When the gRNA targeting cbh1 was used, eight among the twenty seven transformants were found to lose the ability to express CBH1, indicative of successful cbh1 disruption through genome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of these nucleotides, were inserted in the disrupted cbh1 locus.Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei and may find wide applications in strain improvement and functional genomics study.


A copper-controlled RNA interference system for reversible silencing of target genes in Trichoderma reesei.

Researchers incorporated the copper-responsive tcu1 promoter into an RNAi-mediated silencing system to develop a controllable RNAi-mediated silencing system in one sort of fungus, T. reesei. As the proof-of-concept, a prototrophic pyr4 gene, highly expressed cel7a and xyr1 genes induced by Avicel and a fab1 gene, whose knockout has proved to be intractable, were successfully knocked down in the absence of copper when the respective RNAi fragment was expressed. Importantly, the phenotype of RNAi strains was shown to be reversed easily to mimic the complementation for excluding any unwanted effects resulted from the random integration of the hpRNA cassette by adding copper in the media.
we developed an RNAi-mediated silencing system driven by the P tcu1 promoter which is highly responsive to the copper ions. The developed RNAi system could readily knock down/off the target gene in the absence of copper allowing the phenotypical characterization and could mimic the complementation of the deficient strain simply by including copper in the media to exclude the unwanted effect that may result from the random integration of the hpRNA cassette. The copper-responsive RNAi-mediated silencing system is applicable on different nutritional states and represents a powerful tool for characterizing target gene functions in T. reesei.


Reference
1.Rui Liu, Ling Chen, Yanping Jiang, Gen Zou, Zhihua Zhou.A novel transcription factor specifically regulates GH11 xylanase genes in Trichoderma reesei.Biotechnology for Biofuels.2017.10:194
2.Rui Liu,Ling Chen,Yanping Jiang,Zhihua Zhou, Gen Zou,Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system.Cell Discovery.2015.1:15007
3.Zhen zhen Hao, Xiaoyun Su.Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex.BMC Biotechnology.2019.19:2
4.Diego Martinez, Randy M Berka,Bernard Henrissat Thomas,et al.Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina).Nature Biotechnology.2008.26:553-560
5.Lei Wang, Fanglin Zheng, Weixin Zhang et al.Lei Wang, Fanglin Zheng, Weixin Zhang.Biotechnology for Biofuels.2018.11:33

Monday, July 6, 2020

CRISPR Pseudomonas Aeruginosa Services | Gene Knockout, Knockin and Point Mutation, etc.


Pseudomonas aeruginosa is a common encapsulated, Gram-negative, rod-shaped bacterium that can cause disease in plants and animals, including humans. A species of considerable medical importance, P. aeruginosa is a multidrug resistant pathogen recognized for its ubiquity, its intrinsically advanced antibiotic resistance mechanisms, and its association with serious illnesses – hospital-acquired infections such as ventilator-associated pneumonia and various sepsis syndromes. This type of bacteria is found commonly in the environment, like in soil and in water. P. Aeruginosa is a gram-negative, aerobic rod bacterium of the Pseudomonadaceae family (a member of the Gammaproteobacteria). These years, P. aeruginosa are getting more popular in the field of CRISPR editing, which involves gene knockout, gene knock-in and point-mutation in the bacteria.

Applying CRISPR in P. aeruginosa Involving Insertion and Knockout of a Tag Gene
Pseudomonas aeruginosa is both a prototypical multidrug-resistant (MDR) pathogen and a model species for CRISPR-Cas research. The technique is readily applicable in two additional types I-F CRISPR-containing, clinical, and environmental P. aeruginosa isolates. A two-step In-Del strategy involving insertion and subsequent knockout of a tag nearby the desired editing site is further developed to edit the genomic locus lacking an effective PAM (protospacer adjacent motif) or within an essential gene. Among the three resistant mutations synergizing fluoroquinolones resistance, gyrA mutations elicit a greater resistance than drug efflux by MexAB-OprM or MexEF-OprN. These results advanced the understanding of the MDR development of clinical P. aeruginosa strains and demonstrated the great potentials of native CRISPR systems in AMR research. Despite the presence of well-established genetic manipulation tools in various model strains, their applicability in the medically, environmentally, and industrially significant, “non-model” strains is often hampered owing to the vast diversity of DNA homeostasis in these strains and the cytotoxicity of the heterologous CRISPR-Cas9/Cpf1 system. Harnessing the native CRISPR-Cas systems broadly distributed in prokaryotes with built-in genome targeting activity presents a promising and effective approach to resolve these obstacles. The successful development of the first type I-F CRISPR-mediated genome editing technique and its subsequent extension to additional clinical and environmental P. aeruginosa isolates opened a new avenue to the functional genomics of antimicrobial resistance in pathogens.

Silencing and Point Mutations in P. aeruginosa Helps Research in Bacterial Physiology, Drug Target Exploration, and Metabolic Engineering
Pseudomonas species exhibit significant biomedical, ecological, and industrial importance. Despite the extensive research and wide applications, genetic manipulation in Pseudomonas species, in particular in the major human pathogen Pseudomonas aeruginosa, remains a laborious endeavor. It is reported that a genome-editing method pCasPA/pACRISPR was developed by harnessing the CRISPR/Cas9 and the phage λ-Red recombination systems. The method allows for efficient and scarless genetic manipulation in P. aeruginosa. By engineering the fusion of the cytidine deaminase APOBEC1 and the Cas9 nickase, a base editing system pnCasPA-BEC was developed, which enables highly efficient gene inactivation and point mutations in a variety of Pseudomonas species, such as P. aeruginosa. Application of the two genome editing methods will dramatically accelerate a wide variety of investigations, such as bacterial physiology study, drug target exploration, and metabolic engineering.

The Rapid Construction of Gene Knockouts in P. aeruginosa with Base-pair Precision
Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune-compromised patients. As such P. aeruginosa is one of the most well-studied organisms in terms of its genetics. However, the construction of gene KOs and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with the insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum of two weeks. Hence, a rapid streamlined method was developed to create clean KO mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting knockout plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and knockout mutants are identified via sacB mediated sucrose counter-selection. The current method was employed to generate clean gene knockouts of the heme assimilation system anti-σ factor, hasS, and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene knockout was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI knockouts, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly knockout mutants were constructed with single base-pair precision to generate the respective vreA and vreI knockouts while maintaining the start and stop codon of the respective genes. Overall, this method allows for the rapid construction of gene KOs in P. aeruginosa with base-pair precision.

References:
Zeling Xu, Ming Li, Yanran Li, Huiluo Cao, Hua Xiang, Aixin Yan. Native CRISPR-Cas mediated in situ genome editing reveals extensive resistance synergy in the clinical multidrug resistant Pseudomonas aeruginosa. bioRxiv 496711.
Weizhong Chen, Ya Zhang, Yifei Zhang, Yishuang Pi, Tongnian Gu, Liqiang Song, Yu Wang, Quan jiangJi. iScience, Volume 6, 31 August 2018, Pages 222-231.
Huang, Weiliang, and Angela Wilks. “A rapid seamless method for gene KO in Pseudomonas aeruginosa.” BMC microbiology vol. 17,1 199. 19 Sep. 2017, doi:10.1186/s12866-017-1112-5.


Sunday, July 5, 2020

Fungus Genome-editing Services including Knockout, Knock-in, etc.

Fungus, plural fungi, any of about 144,000 known species of organisms of the kingdom Fungi, which includes the yeasts, rusts, smuts, mildews, molds, and mushrooms. There are also many funguslike organisms, including slime molds and oomycetes (water molds), that do not belong to kingdom Fungi but are often called fungi. Many of these fungus-like organisms are included in the kingdom Chromista. Fungi are among the most widely distributed organisms on Earth and are of great environmental and medical importance. Many fungi are free-living in soil or water; others form parasitic or symbiotic relationships with plants or animals.

Fungal pathogens are the main factors responsible for the most severe diseases affecting plants, leading to a significant reduction in yield and crop quality and causing enormous economic losses worldwide. It is estimated that around 30% of the emerging diseases are caused by fungi (Giraud et al., 2010) thus requiring new strategies to improve their management. The arrival of the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR Associated protein 9) genome-editing technique (including gene knockout, knock-in, point-mutation, etc.) enabled researchers to modify genomic sequences in a more precise way. The use of CRISPR-Cas not only provides a time-saving path to perform genomic functional analyses but also could provide new fungal genotypes, that can be used as potential competitors of plant pathogens and/or in the priming of plant defense responses. It is possible to induce the activation of unknown clusters in beneficial fungi by using CRISPR-Cas9, allowing the discovery of new secondary metabolites that could interact with plants or phytopathogens. This could result in new interesting biocontrol strains to be released in the field avoiding the introduction of transgenes in the environment.

 

Applying CRISPR/Cas9 in Yeasts: a Helpful Genetic Changes Tool

The advancement that is driving the rate of adoption of CRISPR–Cas9 for many organisms is its capability of targeting a specific site in a genome: by and large, trying to manipulate a piece of DNA using a homologous fragment of DNA is highly inefficient. One exception to this occurs in the yeast Saccharomyces cerevisiae, where gene editing can be achieved by transforming into cells fewer than 40 nucleotides of sequence divided on either side of a selectable marker. The high proportion of homologous targeting of exogenous DNA into the S. cerevisiae genome is one reason why yeast is such a powerful experimental tool. Indeed, relatively soon after its genome sequence was completed, every gene in the organism was deleted and those sets of mutants made available to researchers. Likewise, by using homologous integration all proteins were tagged with a green fluorescent protein to understand subcellular protein localizations and epitope-tagged to facilitate the global analysis of protein complexes. Thus, this gene-editing system is already considerably beyond just a tool for making genetic changes.

 

Control the Incidence of FHB by CRISPR/Cas9 Silenced Mutants in Fusarium

One possible scenario for the application of CRISPR-Cas9 silenced mutants could be Fusarium Head Blight (FHB), one of the most destructive diseases of grain cereal crops worldwide caused by different Fusarium spp., with F. graminearum and F. culmorum as the most common and aggressive agents. In FHB, while yield loss derives from the sterility of infected florets, grain quality reduction is mainly due to the accumulation of trichothecenes—coded by the fungal tri genes cluster—highly toxic for humans and animals. Previous studies reported that iRNA (interference RNA) Δtri6 mutants of F. culmorum showed reduced disease indices ranging from 40 to 80% on durum wheat (Scherm et al., 2011). Besides, classic knocked-out Δtri5 and Δtri6 mutants of F. graminearum were unable to spread the disease to the adjacent spikelets and grains on wheat and corn, respectively, and also induced plant defense responses (Ravensdale et al., 2014). Likewise, Δmap1 mutants of F. graminearum showed a two-fold reduction of mycotoxin production and were unable to produce perithecia as well as to penetrate in wheat tissues, while the ability to colonize the straw was not affected (Urban et al., 2003). Considering that competition for space and nutrients between virulent and non-virulent strains could reduce the disease, the field release of non-virulent CRISPR-mutant strains of F. graminearum and F. culmorum might help to control the incidence of FHB.

 

Gene knockout Accelerate Functional Genomic Studies in P. chlamydosporia

Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150 μg ml1 G418 sulfate, representing a new selectable marker for P. chlamydosporia. Researchers knocked out one chitinase gene, VFPPC_01099, and two protease genes (VFPPC_10088, VFPPC_06535). Afterward, they obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC_01099), 13% (VFPPC_10088), and 15% (VFPPC_06535). This efficient and convenient technique will accelerate functional genomic studies in P. chlamydosporia.

 

Reference:

Muñoz Isabel Vicente, Sarrocco Sabrina, Malfatti Luca, Baroncelli Riccardo, Vannacci Giovanni. CRISPR-Cas for Fungal Genome Editing: A New Tool for the Management of Plant Diseases. Frontiers in Plant Science. Volume 10, 2019, 135. ISSN: 1664-462X.

Idnurm, A., Meyer, V. The CRISPR revolution in fungal biology and biotechnology, and beyond. Fungal Biol Biotechnol 5, 19 (2018).

Scherm B, Orrù M, Balmas V, Spanu F, Azara E, Delogu G, Hammond TM, Keller NP, Migheli Q. Altered trichothecene biosynthesis in TRI6-silenced transformants of Fusarium culmorum influences the severity of crown and foot rot on durum wheat seedlings. Mol Plant Pathol. 2011 Oct; 12(8):759-71.

Ravensdale M, Rocheleau H, Wang L, Nasmith C, Ouellet T, Subramaniam R. Components of priming-induced resistance to Fusarium head blight in wheat revealed by two distinct mutants of Fusarium graminearum. Mol Plant Pathol. 2014 Dec; 15(9):948-56.

Urban M, Mott E, Farley T, Hammond-Kosack K. The Fusarium graminearum MAP1 gene is essential for pathogenicity and development of perithecia. Mol Plant Pathol. 2003 Sep 1; 4(5):347-59.

Baoming Shen, Jiling Xiao, Liangying Dai, Yonghong Huang, Zhenchuan Mao, Runmao Lin, Yurong Yao, Bingyan Xie. Development of a high-efficiency gene knockout system for Pochonia chlamydosporia. Microbiological Research. Volume 170. January 2015. Page 18-26.

 

 

Thursday, July 2, 2020

A Legendary Life of THP-1 Cell Line









CRISPR Editing Aspergillus nidulans with High Efficiency and Target Rate

Aspergillus nidulans, also called Emericella nidulans, is one of the critical fungal systems in genetics and cell biology. It has been an important research organism for studying eukaryotic cell biology and a wide range of subjects including recombination,DNA repair,mutation, cellcycle control, tubulin, chromatin, nucleokinesis, pathogenesis, metabolism, and experimental evolution.Fungi play a major role in recirculating biomass in ecosystems, as they degrade all types of organic matter. For this reason, they serve as a major source of industrially relevant enzymes, e.g. amylases, cellulases, lipases, pectinases, and proteases. The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. Therefore, to developing versatile methods that can be used to genetically manipulate non-model filamentous fungi, scientists have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. CRISPR technology has revolutionized fungal genetic engineering by increasing the speed and complexity of the experiments that can be performed. Moreover, the efficiency of the system often allows genetic engineering to be introduced in non-model species. Aspergillus nidulans (Emericella nidulans) have a long and productive history as a source of industrial chemicals and enzymes and as a developmental model system to study genetic regulation, developmental biology, signal transduction, and secondary metabolism. So it will be a new pathway to study Aspergillus nidulans by conducting gene knockouts, gene knock-in or point mutations.

Applying CRISPR in highly-efficient marker-free gene targeting in Aspergillus nidulans
The survival of specific Cas9/sgRNA mediated DNA double-strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool, TAPE, to assess protospacer efficiency in Aspergillus nidulans. Moreover, in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, it was showed in the study that even single-stranded oligonucleotides efficiently work as repair templates of specific Cas9/sgRNA induced DNA DSBs in A. nidulans, A. niger, indicating that this type of repair may be wide-spread in filamentous fungi. Importantly, by using single-stranded oligonucleotides for CRISPR-Cas9 mediated gene editing, it is possible to introduce specific point mutations as well gene deletions (gene knockout) at efficiencies approaching 100%. Therefore, it is possible to introduce two-point mutations and one-gene insertion in one transformation experiment with very high efficiency.

Cpf1 enables fast and efficient genome editing in Aspergilli
The efficiency of CRISPR gene editing is due to the formation of specific DNA double-strand breaks made by RNA guided nucleases. In filamentous fungi, only Cas9 has so far been used as the CRISPR nuclease. Since gene editing with Cas9 is limited by its 5′-NGG-3′ protospacer adjacent motif (PAM) sequence, it is important to introduce RNA guided nucleases that depend on other PAM sequences to target a larger repertoire of genomic sites. Cpf1 from Lachnospiraceae bacterium employs a PAM sequence composed of 5′-TTTN-3′ and therefore serves as an attractive option towards this goal. In this study, the Lb_cpf1 codon-optimized for Aspergillus nidulans can be used for CRISPR based gene editing in filamentous fungi. Researchers developed a vector-based setup for Cpf1-mediated CRISPR experiments and showed that it works efficiently at different loci in A. nidulans and A. niger. Specifically, the Cpf1 can catalyze oligonucleotide-mediated genomic site-directed mutagenesis and marker-free gene targeting. The results showed that Cpf1 can be efficiently used in Aspergilli for gene editing thereby expanding the range of genomic DNA sequences that can be targeted by CRISPR technologies.

Efficient CRISPR knockout in A. nidulans
Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. Researchers identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end-joining of DNA in double-strand break repair. Deletion of nkuA (nkuAΔ) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. The selectable heterologous markers were also developed in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuAΔ and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genome-wide gene-targeting projects in A. nidulans feasible.

Ubigene developed CRISPR-B™  which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are much higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Easily achieve microbial gene knockout (KO), point mutation (PM), and knockin (KI).

References:
1. Nødvig CS, Hoof JB, Kogle ME, et al. Efficient oligonucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genet Biol. 2018;115:78-89.
2.  Nødvig CS, Nielsen JB, Kogle ME, Mortensen, UH. A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS One. 2015;10(7):e0133085. Published 2015 Jul 15.
3. Vanegas, K.G., Jarczynska, Z.D., Strucko, T. et al. Cpf1 enables fast and efficient genome editing in Aspergilli. Fungal Biol Biotechnol 6, 6 (2019).
4. Nayak T, Szewczyk E, Oakley CE, et al. A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics. 2006;172(3):1557-1566. 

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