-
Nature Oct 2017RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown...
RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.
Topics: Biocatalysis; CRISPR-Associated Proteins; CRISPR-Cas Systems; Cell Line, Tumor; Cell Survival; Escherichia coli; Gene Editing; Gene Knockdown Techniques; Genes, Reporter; HEK293 Cells; Humans; Leptotrichia; Plant Cells; RNA; RNA Interference; Stress, Physiological; Substrate Specificity
PubMed: 28976959
DOI: 10.1038/nature24049 -
Clinical Microbiology Reviews Apr 2016Bacterial vaginosis (BV) is the most commonly reported microbiological syndrome among women of childbearing age. BV is characterized by a shift in the vaginal flora from... (Review)
Review
Bacterial vaginosis (BV) is the most commonly reported microbiological syndrome among women of childbearing age. BV is characterized by a shift in the vaginal flora from the dominant Lactobacillus to a polymicrobial flora. BV has been associated with a wide array of health issues, including preterm births, pelvic inflammatory disease, increased susceptibility to HIV infection, and other chronic health problems. A number of potential microbial pathogens, singly and in combinations, have been implicated in the disease process. The list of possible agents continues to expand and includes members of a number of genera, including Gardnerella, Atopobium, Prevotella, Peptostreptococcus, Mobiluncus, Sneathia, Leptotrichia, Mycoplasma, and BV-associated bacterium 1 (BVAB1) to BVAB3. Efforts to characterize BV using epidemiological, microscopic, microbiological culture, and sequenced-based methods have all failed to reveal an etiology that can be consistently documented in all women with BV. A careful analysis of the available data suggests that what we term BV is, in fact, a set of common clinical signs and symptoms that can be provoked by a plethora of bacterial species with proinflammatory characteristics, coupled to an immune response driven by variability in host immune function.
Topics: Bacteria; DNA, Bacterial; Female; Humans; Microbiota; Vaginosis, Bacterial
PubMed: 26864580
DOI: 10.1128/CMR.00075-15 -
Nature Protocols Oct 2019Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform...
Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.
Topics: CRISPR-Cas Systems; DNA Primers; Endonucleases; Humans; Leptotrichia; Nucleic Acid Amplification Techniques; Nucleic Acids; Protein Engineering; RNA, Guide, CRISPR-Cas Systems; Recombinant Proteins; Ribonucleases; Workflow; Zika Virus; Zika Virus Infection
PubMed: 31548639
DOI: 10.1038/s41596-019-0210-2 -
Journal of Dental Research Feb 2019Periodontal diseases are initiated by bacteria that accumulate in a biofilm on the tooth surface and affect the adjacent periodontal tissue. Systemic diseases such as... (Review)
Review
Periodontal diseases are initiated by bacteria that accumulate in a biofilm on the tooth surface and affect the adjacent periodontal tissue. Systemic diseases such as diabetes, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) increase susceptibility to destructive periodontal diseases. In human studies and in animal models, these diseases have been shown to enhance inflammation in the periodontium and increase the risk or severity of periodontitis. All 3 systemic diseases are linked to a decrease in bacterial taxa associated with health and an increase in taxa associated with disease. Although there is controversy regarding the specific oral bacterial changes associated with each disease, it has been reported that diabetes increases the levels of Capnocytophaga, Porphyromonas, and Pseudomonas, while Prevotella and Selenomonas are increased in RA and Selenomonas, Leptotrichia, and Prevotella in SLE. In an animal model, diabetes increased the pathogenicity of the oral microbiome, as shown by increased inflammation, osteoclastogenesis, and periodontal bone loss when transferred to normal germ-free hosts. Moreover, in diabetic animals, the increased pathogenicity could be substantially reversed by inhibition of IL-17, indicating that host inflammation altered the microbial pathogenicity. Increased IL-17 has also been shown in SLE, RA, and leukocyte adhesion deficiency and may contribute to oral microbial changes in these diseases. Successful RA treatment with anti-inflammatory drugs partially reverses the oral microbial dysbiosis. Together, these data demonstrate that systemic diseases characterized by enhanced inflammation disturb the oral microbiota and point to IL-17 as key mediator in this process.
Topics: Animals; Bacteria; Cardiovascular Diseases; Diabetes Mellitus, Type 2; Dysbiosis; Humans; Microbiota; Mouth; Periodontitis; Periodontium
PubMed: 30359170
DOI: 10.1177/0022034518805739 -
Science (New York, N.Y.) Aug 2016The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated genes (Cas) adaptive immune system defends microbes against foreign genetic...
The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated genes (Cas) adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the class 2 type VI CRISPR-Cas effector C2c2 and demonstrate its RNA-guided ribonuclease function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis shows that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave single-stranded RNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, mutations of which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.
Topics: Adaptive Immunity; Bacterial Proteins; Base Sequence; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Endoribonucleases; Escherichia coli; Gene Knockdown Techniques; Genetic Loci; Leptotrichia; Levivirus; Molecular Sequence Data; Protein Structure, Tertiary; RNA Cleavage; RNA, Guide, CRISPR-Cas Systems
PubMed: 27256883
DOI: 10.1126/science.aaf5573 -
Microbiology Spectrum Oct 2022As the fourth most common gynecological cancer, cervical cancer has resulted in more than 300,000 deaths worldwide in 2020. The expression of the human papillomavirus...
As the fourth most common gynecological cancer, cervical cancer has resulted in more than 300,000 deaths worldwide in 2020. The expression of the human papillomavirus (HPV) oncogenes E6 and E7 is significantly involved in the initiation and progression of cervical neoplasia. Additionally, the composition of the vaginal microbiome was also closely associated with the ability of HPV to induce cervical cancer. However, the relationship between the expression of HPV E6/E7 oncogene and the composition of the vaginal microbiome has not been clearly explored. In our present study, to investigate the relationship between the HPV E6/E7 oncogene and vaginal microbiome, cervical swabs from 115 patients were collected, and their vaginal microbiomes were analyzed by using metagenomics sequencing. Along with the progression of cervical lesions, the diversity of cervical flora increased gradually, and the abundance of decreased. Compared with the HPV group, the prevalence of HPV E6/E7 and oncogene expression level were significantly upregulated in cervical intraepithelial neoplasia (CIN) and cervical cancer (CC) groups. Additionally, a positive correlation between the expression of the HPV oncogene and the genera , Salmonella, , Pseudomonas, and in the HPV group was observed. In the CIN group, the enrichment of the genera and was weakly linked with HPV oncogene overexpression. In the CC group, a strong association between the overabundance of the genera and and the high expression of HPV oncogene was also found. The increased diversity of the vaginal microbiota and the decreased abundance were significantly associated with the severity of cervical disease, and the expression of the HPV oncogene could also be regulated by certain pathogens in different stages of cervical lesions. The main findings of this study were that we clarified the associations of the different bacterial species with the expression of human papillomavirus (HPV) oncogenes at different stages of cervical cancer. Along with the severity of cervical lesions, the abundance of the genus and species of obviously declined, while the aerobic and anaerobic bacteria, as well as the prevalence and expression of HPV E6/E7 oncogene, were increased dramatically.
Topics: Female; Humans; Papillomaviridae; Uterine Cervical Neoplasms; Papillomavirus Infections; Alphapapillomavirus; Oncogene Proteins, Viral; Dysbiosis; Papillomavirus E7 Proteins; Oncogenes; Uterine Cervical Dysplasia
PubMed: 36036584
DOI: 10.1128/spectrum.00151-22 -
Cell Aug 2017Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To...
Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.
Topics: Bacterial Proteins; Base Sequence; CRISPR-Associated Proteins; CRISPR-Cas Systems; Leptotrichia; Models, Molecular; RNA Processing, Post-Transcriptional; RNA, Bacterial; RNA, Guide, CRISPR-Cas Systems; RNA, Viral; X-Ray Diffraction
PubMed: 28757251
DOI: 10.1016/j.cell.2017.06.050 -
Frontiers in Immunology 2022() eradication has been reported to cause short-term disruption of gut microbiota. It is acknowledged that probiotics supplementation mitigates side effects induced by... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
() eradication has been reported to cause short-term disruption of gut microbiota. It is acknowledged that probiotics supplementation mitigates side effects induced by eradication, yet its role on alleviating dysbiosis of microbiota is obscure.
OBJECTIVES
To evaluate the impact of probiotics on gastrointestinal microbiota after eradication therapy.
METHODS
This was a multicenter, double-blinded, randomized trial done at seven centers in China. A total of 276 treatment-naïve -positive patients were randomly assigned to receive 14-day bismuth-containing quadruple therapy (esomeprazole, bismuth, amoxicillin, furazolidone) combined with probiotics (Bifidobacterium Tetragenous viable Bacteria Tablets) (n=140) or placebo (n=136) for 28 days. Saliva, gastric mucosa and fecal samples were collected before and after therapy for 16S rRNA gene sequencing.
RESULTS
The incidence of gastrointestinal adverse events was lower in probiotics group compared to placebo group (23.6% vs 37.7%, p=0.016), while there was no significant difference in eradication rate. We found dramatic perturbations of gut microbiota immediately following eradication, with the predominance of Proteobacteria in replacement of commensal Firmicutes and Bacteroidetes, and gradually restored after two weeks. The reduction of gut Bacteroidetes caused by eradication drugs was neutralized with probiotics supplementation. The gastric microbiota was completely reconstituted with depleted and other taxa flourished. Of note, patients treated with probiotics showed smaller fluctuations of gastric microbiota compared to those with placebo. We also observed changes of saliva microbiota after eradication, illustrated by the overgrowth of and depletion of . The expansion of some pathogenic genera, including , , in the mouth was suppressed by probiotics.
CONCLUSION
This study not only demonstrated the beneficial effect of probiotics implementation on side events during eradication but also provided a comprehensive profile of microbiome alterations along gastrointestinal tract that modulated by probiotics.
Topics: Humans; Helicobacter pylori; Gastrointestinal Microbiome; Helicobacter Infections; Bismuth; RNA, Ribosomal, 16S; Anti-Bacterial Agents; Probiotics; Bacteroidetes
PubMed: 36426355
DOI: 10.3389/fimmu.2022.1033063 -
Gut Jan 2018A history of periodontal disease and the presence of circulating antibodies to selected oral pathogens have been associated with increased risk of pancreatic cancer;...
OBJECTIVE
A history of periodontal disease and the presence of circulating antibodies to selected oral pathogens have been associated with increased risk of pancreatic cancer; however, direct relationships of oral microbes with pancreatic cancer have not been evaluated in prospective studies. We examine the relationship of oral microbiota with subsequent risk of pancreatic cancer in a large nested case-control study.
DESIGN
We selected 361 incident adenocarcinoma of pancreas and 371 matched controls from two prospective cohort studies, the American Cancer Society Cancer Prevention Study II and the National Cancer Institute Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. From pre-diagnostic oral wash samples, we characterised the composition of the oral microbiota using bacterial 16S ribosomal RNA (16S rRNA) gene sequencing. The associations between oral microbiota and risk of pancreatic cancer, controlling for the random effect of cohorts and other covariates, were examined using traditional and L1-penalised least absolute shrinkage and selection operator logistic regression.
RESULTS
Carriage of oral pathogens, and , were associated with higher risk of pancreatic cancer (adjusted OR for presence vs absence=1.60 and 95% CI 1.15 to 2.22; OR=2.20 and 95% CI 1.16 to 4.18, respectively). Phylum and its genus were associated with decreased pancreatic cancer risk (OR per per cent increase of relative abundance=0.94 and 95% CI 0.89 to 0.99; OR=0.87 and 95% CI 0.79 to 0.95, respectively). Risks related to these phylotypes remained after exclusion of cases that developed within 2 years of sample collection, reducing the likelihood of reverse causation in this prospective study.
CONCLUSIONS
This study provides supportive evidence that oral microbiota may play a role in the aetiology of pancreatic cancer.
Topics: Adenocarcinoma; Aged; Aggregatibacter actinomycetemcomitans; Carrier State; Case-Control Studies; Female; Fusobacteria; High-Throughput Nucleotide Sequencing; Humans; Male; Microbiota; Middle Aged; Mouth; Pancreatic Neoplasms; Periodontium; Porphyromonas gingivalis; Prospective Studies; Risk Factors
PubMed: 27742762
DOI: 10.1136/gutjnl-2016-312580 -
Journal of Oral Microbiology 2017species are non-motile facultative anaerobic/anaerobic bacteria that are found mostly in the oral cavity and some other parts of the human body, in animals, and even in... (Review)
Review
species are non-motile facultative anaerobic/anaerobic bacteria that are found mostly in the oral cavity and some other parts of the human body, in animals, and even in ocean sediments. Valid species include , , , , , , and . Some species require serum or blood for growth. All species ferment carbohydrates and produce lactic acid that may be involved with tooth decay. Acting as opportunistic pathogens, they are involved in a variety of diseases, and have been isolated from immunocompromised but also immunocompetent individuals. Mucositis, oral lesions, wounds, and abscesses may predispose to septicemia. Because identification of species by phenotypic features occasionally lead to misidentification, genetic techniques such as 16S rRNA gene sequencing is recommended. Early diagnosis and treatment of leptotrichia infections is important for positive outcomes. Over the last years, species have been associated with several changes in taxonomy and new associations with clinical diseases. Such changes are reported in this updated review.
PubMed: 29081911
DOI: 10.1080/20002297.2017.1368848