In vitro CRISPR/Cas9 system for genome editing of Aspergillus niger based on removable bidirectional selection marker AmdS

AbstractBiofilm formation on both biotic and abiotic surfaces accounts for a major factor in spread of antimicrobial resistance. Due to their ubiquitous nature, biofilms are of great concern for environment as well as human health. In the present study, an integrated process for the co-production of a cocktail of carbohydrases from a natural variant of Aspergillus niger was designed. The enzyme cocktail was found to have a noteworthy potential to eradicate/disperse the biofilms of selected pathogens. For application of enzymes as an antibiofilm agent, the enzyme productivities were enhanced by statistical modelling using response surface methodology (RSM). The antibiofilm potential of the enzyme cocktail was studied in terms of (i) in vitro cell dispersal assay (ii) release of reducing sugars from the biofilm polysaccharides (iii) the effect of enzyme treatment on biofilm cells and architecture by confocal laser scanning microscopy (CLSM). Potential of the enzyme cocktail to disrupt/disperse the biofilm of selected pathogens from biopolymer surfaces was also assessed by field emission scanning electron microscopy (FESEM) analysis. Further, their usage in conjunction with antibiotics was assessed and it was inferred from the results that the use of enzyme cocktail augmented the efficacy of the antibiotics. The study thus provides promising insights into the prospect of using multiple carbohydrases for management of heterogeneous biofilms formed in natural and clinical settings.

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EFFECTS OF THE ESSENTIAL OIL OF ZATARIA MULTIFLORA BOISS, A THYME-LIKE MEDICINAL PLANT FROM IRAN ON THE GROWTH AND SPORULATION OF ASPERGILLUS NIGER BOTH IN VITRO AND ON LIME FRUITS

Journal of Food Safety ◽

10.1111/j.1745-4565.2011.00317.x ◽

2011 ◽

Vol 31 (3) ◽

pp. 424-432 ◽

Cited By ~ 11

Author(s):

MAHNAZ ABDOLLAHI ◽

HABIBALLAH HAMZEHZARGHANI ◽

MOHAMMAD JAMAL SAHARKHIZ

Keyword(s):

Aspergillus Niger ◽

Essential Oil ◽

Medicinal Plant ◽

Zataria Multiflora

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CRISPR‐Cas9 Genome Editing of Primary Human Vascular Cells In Vitro

Current Protocols ◽

10.1002/cpz1.291 ◽

2021 ◽

Vol 1 (11) ◽

Author(s):

Deepak S. Atri ◽

Vivian S. Lee‐Kim ◽

Shamsudheen K. Vellarikkal ◽

Oscar Sias‐Garcia ◽

Mounica Yanamandala ◽

...

Keyword(s):

Genome Editing ◽

Vascular Cells

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Genome-editing applications of CRISPR–Cas9 to promote in vitro studies of Alzheimer’s disease

Clinical Interventions in Aging ◽

10.2147/cia.s155145 ◽

2018 ◽

Vol Volume 13 ◽

pp. 221-233 ◽

Cited By ~ 15

Author(s):

Vo Van Giau ◽

Hyon Lee ◽

Kyu Hwan Shim ◽

Eva Bagyinszky ◽

Seong Soo A An

Keyword(s):

Genome Editing ◽

In Vitro Studies

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Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

International Journal of Molecular Sciences ◽

10.3390/ijms23020837 ◽

2022 ◽

Vol 23 (2) ◽

pp. 837

Author(s):

Sudip Biswas ◽

Nancy J. Wahl ◽

Michael J. Thomson ◽

John M. Cason ◽

Bill F. McCutchen ◽

...

Keyword(s):

Genome Editing ◽

New Technologies ◽

Fluorescent Protein ◽

Peanut Butter ◽

Processing System ◽

Protoplast Isolation ◽

Sequencing Analysis ◽

Ara H 2

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

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Cyclopeptide Derivatives from the Sponge-Derived Fungus Acremonium persicinum F10

Marine Drugs ◽

10.3390/md19100537 ◽

2021 ◽

Vol 19 (10) ◽

pp. 537

Author(s):

Yingxin Li ◽

Zhiyong Li

Keyword(s):

Amino Acids ◽

Aspergillus Niger ◽

Diffraction Analysis ◽

2D Nmr ◽

Ferric Ions ◽

Single Crystal Diffraction ◽

X Ray ◽

Planar Structures ◽

Anti Fungal

Cyclopeptides usually play a pivotal role, either in the viability or virulence of fungi. Two types of cyclopeptides, six new hydroxamate siderophore cyclohexapeptides (1–6), including acremonpeptides E and F, and their complexes with aluminum and ferric ions; one new cyclic pentapeptolide, aselacin D (9); together with a known compound, aselacin C (10), were isolated and characterized from the sponge-derived fungus Acremonium persicinum F10. In addition, two new siderophore analogues chelating gallium ions (Ga3+), Ga (Ⅲ)-acremonpeptide E (7) and Ga (Ⅲ)-acremonpeptide F (8), using isolated acremonpeptides E and F, were prepared. The planar structures of 1–10 were elucidated by HRESIMS and (1D and 2D) NMR. The absolute configurations of amino acids were determined by means of the advanced Marfey’s method and X-ray single-crystal diffraction analysis. X-ray fluorescence (XRF) spectrometer was performed to disclose the elements of compound 1, indicating the existence of aluminum (Al). Al (Ⅲ)-acremonpeptides E (1), Ga (Ⅲ)-acremonpeptides E (5), Al (Ⅲ)-acremonpeptide F (7), and Ga (Ⅲ)-acremonpeptide F (8) displayed high in vitro anti-fungal activities, which are comparable to amphotericin B, against Aspergillus fumigatus and Aspergillus niger.

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A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽

10.1182/blood-2021-154505 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1862-1862

Author(s):

Gregory J. Cost ◽

Morayma Temoche-Diaz ◽

Janet Mei ◽

Cristina N. Butterfield ◽

Christopher T. Brown ◽

...

Keyword(s):

Amino Acid ◽

Genome Editing ◽

Mammalian Cells ◽

Conflicts Of Interest ◽

Attractive Alternative ◽

Vitro Assay ◽

Crispr System ◽

Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

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Characterizing a thermostable Cas9 for bacterial genome editing and silencing

10.1101/177717 ◽

2017 ◽

Author(s):

Ioannis Mougiakos ◽

Prarthana Mohanraju ◽

Elleke F. Bosma ◽

Valentijn Vrouwe ◽

Max Finger Bou ◽

...

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Gene Deletion ◽

Elevated Temperatures ◽

Bacterial Genome ◽

Thermophilic Bacterium ◽

Geobacillus Thermodenitrificans ◽

Temperature Range ◽

Fundamental Research

AbstractCRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20°C and 70°C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55°C in Bacillus smithii and for gene deletion at 37°C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

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Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA

10.1101/2020.12.21.423781 ◽

2020 ◽

Author(s):

Regina Tkach ◽

Natalia Nikitchina ◽

Nikita Shebanov ◽

Vladimir Mekler ◽

Egor Ulashchik ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Target Recognition ◽

Human Cells ◽

Stable Complex ◽

Slight Effect ◽

Target Dna ◽

Type V

ABSTRACTCRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5’-scaffold moiety and 3’-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases.

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CRISPR and KRAS: a match yet to be made

Journal of Biomedical Science ◽

10.1186/s12929-021-00772-0 ◽

2021 ◽

Vol 28 (1) ◽

Author(s):

Guzide Bender ◽

Rezan Fahrioglu Yamaci ◽

Bahar Taneri

Keyword(s):

Genome Editing ◽

Therapeutic Potential ◽

Treatment Strategies ◽

Tumor Growth Inhibition ◽

Kras Mutations ◽

Main Research ◽

Public Health Burden ◽

Pancreatic Cancers

AbstractCRISPR (clustered regularly interspaced short palindromic repeats) systems are one of the most fascinating tools of the current era in molecular biotechnology. With the ease that they provide in genome editing, CRISPR systems generate broad opportunities for targeting mutations. Specifically in recent years, disease-causing mutations targeted by the CRISPR systems have been of main research interest; particularly for those diseases where there is no current cure, including cancer. KRAS mutations remain untargetable in cancer. Mutations in this oncogene are main drivers in common cancers, including lung, colorectal and pancreatic cancers, which are severe causes of public health burden and mortality worldwide, with no cure at hand. CRISPR systems provide an opportunity for targeting cancer causing mutations. In this review, we highlight the work published on CRISPR applications targeting KRAS mutations directly, as well as CRISPR applications targeting mutations in KRAS-related molecules. In specific, we focus on lung, colorectal and pancreatic cancers. To date, the limited literature on CRISPR applications targeting KRAS, reflect promising results. Namely, direct targeting of mutant KRAS variants using various CRISPR systems resulted in significant decrease in cell viability and proliferation in vitro, as well as tumor growth inhibition in vivo. In addition, the effect of mutant KRAS knockdown, via CRISPR, has been observed to exert regulatory effects on the downstream molecules including PI3K, ERK, Akt, Stat3, and c-myc. Molecules in the KRAS pathway have been subjected to CRISPR applications more often than KRAS itself. The aim of using CRISPR systems in these studies was mainly to analyze the therapeutic potential of possible downstream and upstream effectors of KRAS, as well as to discover further potential molecules. Although there have been molecules identified to have such potential in treatment of KRAS-driven cancers, a substantial amount of effort is still needed to establish treatment strategies based on these discoveries. We conclude that, at this point in time, despite being such a powerful directed genome editing tool, CRISPR remains to be underutilized for targeting KRAS mutations in cancer. Efforts channelled in this direction, might pave the way in solving the long-standing challenge of targeting the KRAS mutations in cancers.

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