scholarly journals The molecular basis for recognition of 5′-NNNCC-3′ PAM and its methylation state by Acidothermus cellulolyticus Cas9

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Anuska Das ◽  
Travis H. Hand ◽  
Chardasia L. Smith ◽  
Ethan Wickline ◽  
Michael Zawrotny ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Methylation Status ◽  
Extreme Environments ◽  
High Sensitivity ◽  
High Specificity ◽  
Acidothermus Cellulolyticus ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Bond Network ◽  
Rna And Dna

AbstractAcidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5′-NNNCC-3′ Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.

scholarly journals Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection

2022 ◽  
Vol 2 (1) ◽  
Author(s):  
Long T. Nguyen ◽  
Santosh R. Rananaware ◽  
Brianna L. M. Pizzano ◽  
Brandon T. Stone ◽  
Piyush K. Jain
Keyword(s):  
High Sensitivity ◽  
High Specificity ◽  
Detection Methods ◽  
Clinical Validation ◽  
Respiratory Pathogens ◽  
Detection Capability ◽  
Guide Rna ◽  
Assay Optimization ◽  
Dual Reporter ◽  
Wide Range

Abstract Background The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics. Methods We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability. Results Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature. Conclusions CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

scholarly journals Radiochemistry, Production Processes, Labeling Methods, and ImmunoPET Imaging Pharmaceuticals of Iodine-124

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 414
Author(s):  
Krishan Kumar ◽  
Arijit Ghosh
Keyword(s):  
Physical Properties ◽  
Imaging Modality ◽  
Target Selection ◽  
Positron Emission Tomography Imaging ◽  
High Sensitivity ◽  
High Specificity ◽  
Production Processes ◽  
Positron Emission ◽  
Significant Research ◽  
Clinical Environments

Target-specific biomolecules, monoclonal antibodies (mAb), proteins, and protein fragments are known to have high specificity and affinity for receptors associated with tumors and other pathological conditions. However, the large biomolecules have relatively intermediate to long circulation half-lives (>day) and tumor localization times. Combining superior target specificity of mAbs and high sensitivity and resolution of the PET (Positron Emission Tomography) imaging technique has created a paradigm-shifting imaging modality, ImmunoPET. In addition to metallic PET radionuclides, 124I is an attractive radionuclide for radiolabeling of mAbs as potential immunoPET imaging pharmaceuticals due to its physical properties (decay characteristics and half-life), easy and routine production by cyclotrons, and well-established methodologies for radioiodination. The objective of this report is to provide a comprehensive review of the physical properties of iodine and iodine radionuclides, production processes of 124I, various 124I-labeling methodologies for large biomolecules, mAbs, and the development of 124I-labeled immunoPET imaging pharmaceuticals for various cancer targets in preclinical and clinical environments. A summary of several production processes, including 123Te(d,n)124I, 124Te(d,2n)124I, 121Sb(α,n)124I, 123Sb(α,3n)124I, 123Sb(3He,2n)124I, natSb(α, xn)124I, natSb(3He,n)124I reactions, a detailed overview of the 124Te(p,n)124I reaction (including target selection, preparation, processing, and recovery of 124I), and a fully automated process that can be scaled up for GMP (Good Manufacturing Practices) production of large quantities of 124I is provided. Direct, using inorganic and organic oxidizing agents and enzyme catalysis, and indirect, using prosthetic groups, 124I-labeling techniques have been discussed. Significant research has been conducted, in more than the last two decades, in the development of 124I-labeled immunoPET imaging pharmaceuticals for target-specific cancer detection. Details of preclinical and clinical evaluations of the potential 124I-labeled immunoPET imaging pharmaceuticals are described here.

scholarly journals Highly Sensitive Fluorescent Detection of Acetylcholine Based on the Enhanced Peroxidase-Like Activity of Histidine Coated Magnetic Nanoparticles

Nanomaterials ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1207
Author(s):  
Hong Jae Cheon ◽  
Quynh Huong Nguyen ◽  
Moon Il Kim
Keyword(s):  
Magnetic Nanoparticles ◽  
High Sensitivity ◽  
High Specificity ◽  
Choline Oxidase ◽  
Fluorescent Detection ◽  
One Pot ◽  
Site Structure ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Peroxidase Mimics

Inspired by the active site structure of natural horseradish peroxidase having iron as a pivotal element with coordinated histidine residues, we have developed histidine coated magnetic nanoparticles (His@MNPs) with relatively uniform and small sizes (less than 10 nm) through one-pot heat treatment. In comparison to pristine MNPs and other amino acid coated MNPs, His@MNPs exhibited a considerably enhanced peroxidase-imitating activity, approaching 10-fold higher in catalytic reactions. With the high activity, His@MNPs then were exploited to detect the important neurotransmitter acetylcholine. By coupling choline oxidase and acetylcholine esterase with His@MNPs as peroxidase mimics, target choline and acetylcholine were successfully detected via fluorescent mode with high specificity and sensitivity with the limits of detection down to 200 and 100 nM, respectively. The diagnostic capability of the method is demonstrated by analyzing acetylcholine in human blood serum. This study thus demonstrates the potential of utilizing His@MNPs as peroxidase-mimicking nanozymes for detecting important biological and clinical targets with high sensitivity and reliability.

Cds nanocrystal enhanced TiO2photoelectrochemical aptasensor for sensitive detection of cytochrome c

2021 ◽  
Vol 11 (11) ◽  
pp. 1774-1780
Author(s):  
Shanji Fan ◽  
Hong Huang ◽  
Hong Chen ◽  
Jiachi Xu ◽  
Zecheng Hu ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Limit Of Detection ◽  
Specific Binding ◽  
High Sensitivity ◽  
High Specificity ◽  
Linear Range ◽  
Current Intensity ◽  
Sensitive Detection ◽  
Layer Adsorption ◽  
Ionic Layer

A CdS nanocrystal enhanced TiO2 nanotubes (CdS@TiO2 NATs) photoelectrode was prepared via successive ionic layer adsorption and reaction (SILAR) of CdS on the surface of TiO2 NATs. A HS-aptamer owing a specific binding toward cytochrome c was modified onto the CdS@TiO2 NATs, which resulting a decrease in the photoelectrical current intensity. Cytochrome c is therefore quantified based on the decrease in photoelectrical current. High specificity and high sensitivity were obtained with a linear range from 3 pM to 80 nM, and a limit of detection of 2.53 pM.

scholarly journals A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections

2003 ◽  
Vol 17 (2) ◽  
pp. 142-146 ◽  
Author(s):  
José Freitas Siqueira Júnior ◽  
Isabela das Neves Rôças
Keyword(s):  
16S Rdna ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Nested Polymerase Chain Reaction ◽  
Oral Infections ◽  
Root Canals ◽  
Infected Root ◽  
Pcr Products ◽  
Species Specific

The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification.

scholarly journals Comparison between near miss criteria in a maternal intensive care unit

2018 ◽  
Vol 52 (0) ◽  
Author(s):  
Alana Santos Monte ◽  
Liana Mara Rocha Teles ◽  
Mônica Oliveira Batista Oriá ◽  
Francisco Herlânio Costa Carvalho ◽  
Helen Brown ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
Maternal Death ◽  
High Sensitivity ◽  
High Specificity ◽  
Near Miss ◽  
World Health ◽  
Maternal Near Miss ◽  
Chi Square ◽  
Cross Sectional

ABSTRACT Objective: The aim of this study was to compare the incidence of different criteria of maternal near miss in women admitted to an obstetric intensive care unit and their sensitivity and specificity in identifying cases that have evolved to morbidity. Method: A cross-sectional analytical epidemiological study was conducted with women admitted to the intensive care unit of the Maternity School Assis Chateaubriand in Ceará, Brazil. The Chi-square test and odds ratio were used. Results: 560 records were analyzed. The incidence of maternal near miss ranged from 20.7 in the Waterstone criteria to 12.4 in the Geller criteria. The maternal near-miss mortality ratio varied from 4.6:1 to 7.1:1, showing better index in the Waterstone criteria, which encompasses a greater spectrum of severity. The Geller and Mantel criteria, however, presented high sensitivity and low specificity. Except for the Waterstone criteria, there was an association between the three other criteria and maternal death. Conclusion: The high specificity of Geller and Mantel criteria in identifying maternal near miss considering the World Health Organization criteria as a gold standard and a lack of association between the criteria of Waterstone with maternal death.

scholarly journals Electroanalytical Biosensors for Circulating Tumor DNA Detection—A Brief Review

2021 ◽  
Author(s):  
Zhijia Peng ◽  
Xiaogang Lin ◽  
Weiqi Nian ◽  
Xiaodong Zheng ◽  
Jayne Wu
Keyword(s):  
Real Time ◽  
Point Of Care ◽  
Low Cost ◽  
High Sensitivity ◽  
High Specificity ◽  
Circulating Tumor Dna ◽  
Minimal Invasive ◽  
Diagnosis And Treatment ◽  
Detection Technology ◽  
Tumor Dna

Early diagnosis and treatment have always been highly desired in the fight against cancer, and detection of circulating tumor DNA (ctDNA) has recently been touted as highly promising for early cancer screening. Consequently, the detection of ctDNA in liquid biopsy gains much attention in the field of tumor diagnosis and treatment, which has also attracted research interest from the industry. However, traditional gene detection technology is difficult to achieve low cost, real-time and portable measurement of ctDNA. Electroanalytical biosensors have many unique advantages such as high sensitivity, high specificity, low cost and good portability. Therefore, this review aims to discuss the latest development of biosensors for minimal-invasive, rapid, and real-time ctDNA detection. Various ctDNA sensors are reviewed with respect to their choices of receptor probes, detection strategies and figures of merit. Aiming at the portable, real-time and non-destructive characteristics of biosensors, we analyze their development in the Internet of Things, point-of-care testing, big data and big health.

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

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