scholarly journals RGEN-SEQ FOR HIGHLY SENSITIVE AMPLIFICATION-FREE SCREEN OF OFF-TARGET SITES OF GENE EDITORS

2021 ◽  
Author(s):  
Alexander Kuzin ◽  
Brendan Redler ◽  
Jaya Onuska ◽  
Alexei Slesarev
Keyword(s):  
Target Detection ◽  
Dna Cleavage ◽  
Detailed Comparison ◽  
Detection Methods ◽  
Guide Rnas ◽  
Coverage Bias ◽  
Biochemical Assays ◽  
Sequencing Method ◽  
Highly Sensitive ◽  
Target Sites

Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, they still leave plenty of room for improvement. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. The method is extremely simple, and it is on a par or even supersedes in sensitivity existing assays without reliance on amplification steps. The latter saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs.

scholarly journals RGEN-seq For Highly Sensitive Amplification-free Screen Of Off-target Sites Of Gene Editors

2021 ◽  
Author(s):  
Alexander Kuzin ◽  
Brendan Redler ◽  
Jaya Onuska ◽  
Alexei Slesarev
Keyword(s):  
Target Detection ◽  
Dna Cleavage ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Detailed Comparison ◽  
Detection Methods ◽  
Guide Rnas ◽  
Coverage Bias ◽  
Biochemical Assays ◽  
Target Sites

Abstract Sensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, significant technical and methodological issues still remain.. Of note, existing methods rely on PCR amplification, tagging, and affinity purification which can introduce bias, contaminants, sample loss through handling, etc. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. Through use of novel sequencing adapters, the RGEN-Seq method saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs.

scholarly journals RGEN-seq for highly sensitive amplification-free screen of off-target sites of gene editors

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Kuzin ◽  
Brendan Redler ◽  
Jaya Onuska ◽  
Alexei Slesarev
Keyword(s):  
Target Detection ◽  
Dna Cleavage ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Detailed Comparison ◽  
Detection Methods ◽  
Guide Rnas ◽  
Coverage Bias ◽  
Biochemical Assays ◽  
Target Sites

AbstractSensitive detection of off-target sites produced by gene editing nucleases is crucial for developing reliable gene therapy platforms. Although several biochemical assays for the characterization of nuclease off-target effects have been recently published, significant technical and methodological issues still remain. Of note, existing methods rely on PCR amplification, tagging, and affinity purification which can introduce bias, contaminants, sample loss through handling, etc. Here we describe a sensitive, PCR-free next-generation sequencing method (RGEN-seq) for unbiased detection of double-stranded breaks generated by RNA-guided CRISPR-Cas9 endonuclease. Through use of novel sequencing adapters, the RGEN-Seq method saves time, simplifies workflow, and removes genomic coverage bias and gaps associated with PCR and/or other enrichment procedures. RGEN-seq is fully compatible with existing off-target detection software; moreover, the unbiased nature of RGEN-seq offers a robust foundation for relating assigned DNA cleavage scores to propensity for off-target mutations in cells. A detailed comparison of RGEN-seq with other off-target detection methods is provided using a previously characterized set of guide RNAs.

scholarly journals Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Seung-Hun Kang ◽  
Wi-jae Lee ◽  
Ju-Hyun An ◽  
Jong-Hee Lee ◽  
Young-Hyun Kim ◽  
...  
Keyword(s):  
Target Detection ◽  
High Sensitivity ◽  
Fold Increase ◽  
Amplicon Sequencing ◽  
Detection Methods ◽  
Detection Techniques ◽  
Amplification Method ◽  
Genome Wide ◽  
Sequencing Method ◽  
Specific Amplification

AbstractCRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.

scholarly journals A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Ziying Hu ◽  
Chengdong Zhang ◽  
Daqi Wang ◽  
Siqi Gao ◽  
Sang-Ging Ong ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Dna Cleavage ◽  
Cleavage Activity ◽  
Highly Sensitive ◽  
Activation Assay ◽  
Target Sites ◽  
Multiple Alternative ◽  
Targeted Deep Sequencing ◽  
Novel Applications ◽  
In Cells

CRISPR/Cas9 nucleases hold great potential for gene therapy, but they frequently induce unwanted off-target cleavage. We previously developed a GFP activation assay for detection of DNA cleavage in cells. Here, we demonstrate two novel applications of this assay. First, we use this assay to confirm off-target cleavage that cannot be detected by targeted deep sequencing in cells before. Second, we use this approach to detect multiple alternative PAMs recognized by SpCas9. These noncanonical PAMs are associated with low cleavage activity, but targets associated with these PAMs must be considered as potential off-target sites. Taken together, the GFP activation assay is a powerful platform for DNA cleavage detection in cells.

Detection by real-time quaking-induced conversion (RT-QuIC), ELISA, and IHC of chronic wasting disease prion in lymph nodes from Pennsylvania white-tailed deer with specific PRNP genotypes

2021 ◽  
pp. 104063872110214
Author(s):  
Deepanker Tewari ◽  
David Steward ◽  
Melinda Fasnacht ◽  
Julia Livengood
Keyword(s):  
Real Time ◽  
Disease Susceptibility ◽  
Chronic Wasting Disease ◽  
Low Frequency ◽  
The United States ◽  
Detection Methods ◽  
Spongiform Encephalopathy ◽  
Wasting Disease ◽  
Diagnostic Laboratory ◽  
Highly Sensitive

Chronic wasting disease (CWD) is a prion-mediated, transmissible disease of cervids, including deer ( Odocoileus spp.), which is characterized by spongiform encephalopathy and death of the prion-infected animals. Official surveillance in the United States using immunohistochemistry (IHC) and ELISA entails the laborious collection of lymphoid and/or brainstem tissue after death. New, highly sensitive prion detection methods, such as real-time quaking-induced conversion (RT-QuIC), have shown promise in detecting abnormal prions from both antemortem and postmortem specimens. We compared RT-QuIC with ELISA and IHC for CWD detection utilizing deer retropharyngeal lymph node (RLN) tissues in a diagnostic laboratory setting. The RLNs were collected postmortem from hunter-harvested animals. RT-QuIC showed 100% sensitivity and specificity for 50 deer RLN (35 positive by both IHC and ELISA, 15 negative) included in our study. All deer were also genotyped for PRNP polymorphism. Most deer were homozygous at codons 95, 96, 116, and 226 (QQ/GG/AA/QQ genotype, with frequency 0.86), which are the codons implicated in disease susceptibility. Heterozygosity was noticed in Pennsylvania deer, albeit at a very low frequency, for codons 95GS (0.06) and 96QH (0.08), but deer with these genotypes were still found to be CWD prion-infected.

scholarly journals TWC-Net: A SAR Ship Detection Using Two-Way Convolution and Multiscale Feature Mapping

2021 ◽  
Vol 13 (13) ◽  
pp. 2558
Author(s):  
Lei Yu ◽  
Haoyu Wu ◽  
Zhi Zhong ◽  
Liying Zheng ◽  
Qiuyue Deng ◽  
...  
Keyword(s):  
Target Detection ◽  
Detection Methods ◽  
Observation System ◽  
Single Shot ◽  
Small Target ◽  
Feature Mapping ◽  
Sar Images ◽  
Two Stage ◽  
Ship Detection ◽  
Deep Feature

Synthetic aperture radar (SAR) is an active earth observation system with a certain surface penetration capability and can be employed to observations all-day and all-weather. Ship detection using SAR is of great significance to maritime safety and port management. With the wide application of in-depth learning in ordinary images and good results, an increasing number of detection algorithms began entering the field of remote sensing images. SAR image has the characteristics of small targets, high noise, and sparse targets. Two-stage detection methods, such as faster regions with convolution neural network (Faster RCNN), have good results when applied to ship target detection based on the SAR graph, but their efficiency is low and their structure requires many computing resources, so they are not suitable for real-time detection. One-stage target detection methods, such as single shot multibox detector (SSD), make up for the shortage of the two-stage algorithm in speed but lack effective use of information from different layers, so it is not as good as the two-stage algorithm in small target detection. We propose the two-way convolution network (TWC-Net) based on a two-way convolution structure and use multiscale feature mapping to process SAR images. The two-way convolution module can effectively extract the feature from SAR images, and the multiscale mapping module can effectively process shallow and deep feature information. TWC-Net can avoid the loss of small target information during the feature extraction, while guaranteeing good perception of a large target by the deep feature map. We tested the performance of our proposed method using a common SAR ship dataset SSDD. The experimental results show that our proposed method has a higher recall rate and precision, and the F-Measure is 93.32%. It has smaller parameters and memory consumption than other methods and is superior to other methods.

A rapid and accurate method for screening T-2 toxin in food and feed using competitive AlphaLISA

2021 ◽  
Vol 368 (6) ◽  
Author(s):  
Liwen Zhang ◽  
Qingyu Lv ◽  
Yuling Zheng ◽  
Xuan Chen ◽  
Decong Kong ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Accurate Method ◽  
Detection Methods ◽  
Cereal Crops ◽  
Detection Range ◽  
Highly Sensitive ◽  
Food And Feed ◽  
Good Repeatability ◽  
Feed Samples ◽  
Better Than

ABSTRACT T-2 is a common mycotoxin contaminating cereal crops. Chronic consumption of food contaminated with T-2 toxin can lead to death, so simple and accurate detection methods in food and feed are necessary. In this paper, we establish a highly sensitive and accurate method for detecting T-2 toxin using AlphaLISA. The system consists of acceptor beads labeled with T-2-bovine serum albumin (BSA), streptavidin-labeled donor beads and biotinylated T-2 antibodies. T-2 in the sample matrix competes with T-2-BSA for antibodies. Adding biotinylated antibodies to the test well followed by T-2 and T-2-BSA acceptor beads yielded a detection range of 0.03–500 ng/mL. The half-maximal inhibitory concentration was 2.28 ng/mL and the coefficient of variation was <10%. In addition, this method had no cross-reaction with other related mycotoxins. This optimized method for extracting T-2 from food and feed samples achieved a recovery rate of approximately 90% in T-2 concentrations as low as 1 ng/mL, better than the performance of a commercial ELISA kit. This competitive AlphaLISA method offers high sensitivity, good specificity, good repeatability and simple operation for detecting T-2 toxin in food and feed.

scholarly journals Bacterial colonies detecting and counting based on enhanced CNN detection method

2021 ◽  
Vol 233 ◽  
pp. 02012
Author(s):  
Shousheng Liu ◽  
Zhigang Gai ◽  
Xu Chai ◽  
Fengxiang Guo ◽  
Mei Zhang ◽  
...  
Keyword(s):  
Target Detection ◽  
Error Detection ◽  
Detection Rate ◽  
Detection Method ◽  
Difficult Problem ◽  
Detection Methods ◽  
Detection Accuracy ◽  
Detection Model ◽  
Confidence Threshold ◽  
Small Targets

Bacterial colonies detecting and counting is tedious and time-consuming work. Fortunately CNN (convolutional neural network) detection methods are effective for target detection. The bacterial colonies are a kind of small targets, which have been a difficult problem in the field of target detection technology. This paper proposes a small target enhancement detection method based on double CNNs, which can not only improve the detection accuracy, but also maintain the detection speed similar to the general detection model. The detection method uses double CNNs. The first CNN uses SSD_MOBILENET_V1 network with both target positioning and target recognition functions. The candidate targets are screened out with a low confidence threshold, which can ensure no missing detection of small targets. The second CNN obtains candidate target regions according to the first round of detection, intercepts image sub-blocks one by one, uses the MOBILENET_V1 network to filter out targets with a higher confidence threshold, which can ensure good detection of small targets. Through the two-round enhancement detection method has been transplanted to the embedded platform NVIDIA Jetson AGX Xavier, the detection accuracy of small targets is significantly improved, and the target error detection rate and missed detection rate are reduced to less than 1%.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

Universal primers for rapid detection of six pospiviroids in Solanaceae plants using one-step RT-PCR and RT-LAMP

Plant Disease ◽  
2021 ◽  
Author(s):  
Yi-Wen Tseng ◽  
Chien-Fu Wu ◽  
Chia-Hwa Lee ◽  
Chung Jan Chang ◽  
Yuh-Kun Chen ◽  
...  
Keyword(s):  
Potato Spindle Tuber Viroid ◽  
Detection Methods ◽  
Universal Primers ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Highly Sensitive ◽  
One Step ◽  
Tomato Chlorotic Dwarf Viroid ◽  
Minimal Concentration ◽  
Solanaceous Plants

A number of viruses and viroids infect solanaceous plants causing severe yield losses. Several seed-borne viroids are currently listed as quarantine pathogens in many countries. Among them, columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd), and tomato planta macho viroid (TPMVd) are of major concerns. The objective of this study was to design and test universal primers that could be used to detect six viroids in solanaceous plants using one-step RT-PCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Results revealed that a pair of degenerate primers could be used in a one-step RT-PCR to amplify six pospiviroids from Solanaceae seeds and plants. Moreover, five primers were designed and used in RT-LAMP to amplify six pospiviroids. The minimal concentration of viroid RNA required for a successful detection varied, ranging from one femtogram to 10 nanograms, depending on the species of viroid and detection method. In general, RT-LAMP was more sensitive than RT-PCR but both assays were rapid and highly sensitive tools to detect six pospiviroids. Detection methods currently in use for these viroids require at least two different sets of primers. The assays developed in this research could facilitate to screen a large number of solanaceous plants and seeds intended for import and export.

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