scholarly journals Efficient Genome Editing by a Miniature CRISPR-AsCas12f1 Nuclease in Bacillus anthracis

Author(s):  
Yanchun Wang ◽  
Shuli Sang ◽  
Xin Zhang ◽  
Haoxia Tao ◽  
Qing Guan ◽  
...  

A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.

2005 ◽  
Vol 134 (2) ◽  
pp. 315-322 ◽  
Author(s):  
M. D. TANRIOVER ◽  
G. S. GUVEN ◽  
D. SEN ◽  
S. UNAL ◽  
O. UZUN

Sepsis continues to have a substantial mortality and morbidity despite advances in the diagnosis and management of this condition. We retrospectively analysed hospital charts of patients diagnosed to have sepsis between January 2002 and June 2003. Demographic characteristics of patients, microbiological findings and predictors of survival were evaluated. Sixty-nine sepsis episodes that occurred in 63 patients were analysed. The most common underlying diseases were hypertension, malignancies and diabetes mellitus. Renal insufficiency, respiratory distress and disseminated intravascular coagulation developed in 52·2, 30·4 and 30·4% of the episodes respectively; 47·7% of the blood cultures yielded an organism. Gram-negative bacteria were the predominant microorganisms (65·9%). Fifty-five patients (87·3%) died. Mechanical ventilation and underlying renal disease were significant determinants of mortality. In conclusion, Gram-negative bacteria remain the major pathogens in sepsis. The mortality remains very high, and a change in the clinical approach to the septic patient should be employed to improve the outcome.


2001 ◽  
Vol 14 (3) ◽  
pp. 426-430 ◽  
Author(s):  
Bruno Dombrecht ◽  
Jos Vanderleyden ◽  
Jan Michiels

The construction of several stable RK2-derived cloning vectors for the analysis of gene expression and function in gram-negative bacteria is reported. Plasmid stability is conferred by the RK2 par locus or by insertion of the spsAB or spsCD symbiotic plasmid stability loci from pNGR234a of Rhizobium sp. NGR234. The vectors carry multiple cloning sites with protection against read-through transcriptional activity of vector sequences. Vector derivatives with the constitutive nptII promoter or a promoter-less gusA gene are suitable for the study of gene function or regulation in bacteria.


1975 ◽  
Vol 75 (1) ◽  
pp. 99-112 ◽  
Author(s):  
R. J. Jones ◽  
E. A. Roe ◽  
R. E. Dyster

SUMMARYThe Limulus test detected endotoxins in the plasma of burned and unburned mice infected with different species of gram-negative bacteria. Individual strains of different species of gram-negative bacteria produced different amounts of endotoxin in the plasma of infected mice. Plasma from mice given lethal infections showed very high concentrations of endotoxin. Low concentrations of endotoxin in the plasma were tolerated by mice but high concentrations were invariably fatal. A polyvalent pseudomonas vaccine reduced endotoxin in the plasma of mice given lethal infections of Pseudomonas aeruginosa.


2015 ◽  
Vol 64 (2) ◽  
pp. 185-188 ◽  
Author(s):  
GRAMMATO EVANGELOPOULOU ◽  
GEORGIOS FILIOUSSIS ◽  
SPYRIDON KRITAS ◽  
MARIA KANTERE ◽  
ANGELIKI R. BURRIEL

The presence of Gram-negative bacteria species, other than Salmonella spp., in the gallbladder of pigs was examined. Isolated Gram-negative bacteria were assigned to species using the Microgen™ GnA+B-ID Systems. Of the 64 isolated strains 43 were identified as Escherichia coli, seven as Enterobacter spp., three each as Klebsiella spp., Citrobacterfreundii, Aeromonas hydrophila and Cronobacter sakazakii and one each as Escherichiafergusonii and Trabulsiella guamensis. Their antibiograms showed very high resistance to ampicillin, amoxicillin, tetracycline, chloramphenicol and sulfamethoxazole/trimethoprim. It was concluded that the pigs' gallbladder is a reservoir of potentially pathogenic Gram-negative bacteria for pork consumers.


2017 ◽  
Vol 9 (1) ◽  
pp. 1
Author(s):  
Anna Meiliana ◽  
Nurrani Mustika Dewi ◽  
Andi Wijaya

BACKGROUND: Recently established genome editing technologies will open new avenues for biological research and development. Human genome editing is a powerful tool which offers great scientific and therapeutic potential.CONTENT: Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9) technology is revolutionizing the gene function studies and possibly will give rise to an entirely new degree of therapeutics for a large range of diseases. Prompt advances in the CRISPR/Cas9 technology, as well as delivery modalities for gene therapy applications, are dismissing the barriers to the clinical translation of this technology. Many studies conducted showed promising results, but as current available technologies for evaluating off-target gene modification, several elements must be addressed to validate the safety of the CRISPR/Cas9 platform for clinical application, as the ethical implication as well.SUMMARY: The CRISPR/Cas9 system is a powerful genome editing technology with the potential to create a variety of novel therapeutics for a range of diseases, many of which are currently untreatable.KEYWORDS: genome editing, CRISPR-Cas, guideRNA, DSB, ZFNs, TALEN


2006 ◽  
Vol 72 (7) ◽  
pp. 5027-5036 ◽  
Author(s):  
Robert M. Q. Shanks ◽  
Nicky C. Caiazza ◽  
Shannon M. Hinsa ◽  
Christine M. Toutain ◽  
George A. O'Toole

ABSTRACT A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.


2016 ◽  
Vol 14 (34) ◽  
pp. 8047-8052 ◽  
Author(s):  
Ivana Di Bari ◽  
Roberta Picciotto ◽  
Giuseppe Granata ◽  
Anna R. Blanco ◽  
Grazia M. L. Consoli ◽  
...  

Visible light excitation of a calix[4]arene-based nanoconstruct encapsulating a hydrophobic NO releaser triggers NO generation with high efficiency, resulting in a considerable antibacterial activity against both Gram positive and Gram negative bacteria strains.


2019 ◽  
Author(s):  
Willy Klöckner ◽  
Cristian Patiño Vidal ◽  
Carol López de Dicastillo ◽  
Ram Manohar Yadav ◽  
D I N E S H P SINGH

Self-assembled 3D structures with 0, 1 or 2D nano building blocks are fascinating due to its astounding new and enhanced properties for various applications. Here we report the acetonitrile mediated synthesis of self-assembled bulk cylinder of length ~ 9.00 cm and diameter ~ 4.0 cm consolidated with ultralong and very thin silver vanadate nanowires and its enhanced antibacterial activity.  The addition of acetonitrile during synthesis and its volumetric concentration plays an important role in the self-assembly. The use of 30 mL acetonitrile during the mixing of the reactants AgNO3 and NH4VO3 and post hydrothermal treatment at 200 °C for 12 h result the formation of bulk cylinder like structure.  X-ray diffraction (XRD) and Scanning electron microscopy (SEM) revealed that the bulk cylinder is consolidated with ultrathin and long β-AgVO3 nanowires having minimum diameter ~16 nm and of undefined length (approximately 10 mm long). The self-assembled β-AgVO3nanowire like structure with very high surface area exhibited very high antibacterial activity against Listeria innocuaATCC 33090 and Staphylococcus aureusATCC 25923 and Escherichia coliATCC 25922, as Gram-positive and Gram-negative bacteria, respectively. 


2021 ◽  
Author(s):  
Elena Velazquez ◽  
Yamal Al-ramahi ◽  
Jonathan Tellechea-Luzardo ◽  
Natalio Krasnogor ◽  
Victor de Lorenzo

Genome editing methods based on Group II introns (known as Targetron technology) have been long used as a gene knock-out strategy in a wide range of organisms in a fashion independent of homologous recombination. Yet, their utility as delivery systems has been typically suboptimal because of their reduced efficiency of insertion when they carry exogenous sequences. We show that this limitation can be tackled and Targetron adapted as a general tool in Gram-negative bacteria. To this end, a set of broad host range standardized vectors were designed for conditional expression of the Ll.LtrB intron. After testing the correct functionality of these plasmids in Escherichia coli and Pseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. By following this pipeline, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-type P. putida KT2440 and its ∆recA derivative. Finally, we were able to apply this technology to successfully tag this strain with an orthogonal short sequence (barcode) that acts as a unique identifier for tracking this microorganism in biotechnological settings. The results with P. putida exemplified the value of the Targetron approach for unrestricted delivery of small DNA fragments to the genomes of Gram-negative bacteria for a suite of genome editing endeavours.


MediAl ◽  
2019 ◽  
pp. 70-73
Author(s):  
N. A. Gordinskaya ◽  
E. V. Sabirova ◽  
N. V. Abramova

Introduction. The most common mechanism for the resistance of gram-negative bacteria to various classes of antimicrobial agents, including carbapenems, is the production of beta-lactamases, enzymes that destroy the beta-lactam ring. Purpose of the study. To analyze the activity of ceftazidime/avibactam against pseudomonads and Klebsiella isolated in patients with severe thermal injury. Materials and methods. We analyzed 2553 isolates – pathogens of wound burn infection in patients with thermal injury treated in 2018–2019. Results and discussion. Phenotypically, 72,8% of the analyzed P. aeruginosa were resistant to carbapenems, while 56,3% of carbapenemresistant strains produce group Vim metal-beta-lactamases. Analysis of the effectiveness of ceftazidime/avibactam against P. aeruginosa showed its high efficiency, more than half of the strains (55,3%) were sensitive to the drug. The studied K. pneumoniae phenotypically in 63,1% were carbapenem-resistant. Among K. pneumoniae resistant to carbapenems, 89,3% of the strains revealed genes of serine KPC or OXA-48 like carbapenemases. In vitro ceftazidime/avibactam was active against two-thirds (72,7%) of K. pneumoniae strains. Сonclusions. 1. Gram-negative microorganisms occupy 30,2% of the etiological structure of a wound burn infection. 2. Phenotypically 72,8% of Pseudomonas aeruginosa are resistant to carbapenems, 56,3% of them produce metal beta-lactamases. 3. 63,1% of Klebsiella pneumoniae isolated in patients with thermal injury are resistant to carbapenems, 89,3% of them carry cattle or OXA-48 genes like carbapenemases. 4. Ceftazidime / avibactam in vitro showed activity against P. aeruginosa and K. pneumoniae, with 55,3% and 72,7% of the strains, respectively, being sensitive.


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