scholarly journals One-step assembly of large CRISPR arrays enables multi-functional targeting and reveals constraints on array design

2018 ◽  
Author(s):  
Chunyu Liao ◽  
Fani Ttofali ◽  
Rebecca A. Slotkowski ◽  
Steven R. Denny ◽  
Taylor D. Cecil ◽  
...  
Keyword(s):  
Rna Cleavage ◽  
Critical Factors ◽  
Array Design ◽  
Guide Rnas ◽  
Dna Targeting ◽  
Repeat Sequences ◽  
Factors Influencing ◽  
Target Sites ◽  
One Step ◽  
Single Effector

SUMMARYCRISPR-Cas systems inherently multiplex through their CRISPR arrays--whether to confer immunity against multiple invaders or by mediating multi-target editing, regulation, imaging, and sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report an efficient, one-step scheme called CRATES to construct large CRISPR arrays through defined assembly junctions within the trimmed portion of array spacers. We show that the constructed arrays function with the single-effector nucleases Cas9, Cas12a, and Cas13a for multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. We also applied CRATES to assemble composite arrays utilized by multiple Cas nucleases, where these arrays enhanced DNA targeting specificity by blocking off-target sites. Finally, array characterization revealed context-dependent loss of spacer activity and processing of unintended guide RNAs derived from Cas12a terminal repeats. CRATES thus can facilitate diverse applications requiring CRISPR multiplexing and help elucidate critical factors influencing array function.

scholarly journals Evaluating Critical Factors Influencing the Reliability of Emergency Logistics Systems Using Multiple-Attribute Decision Making

Symmetry ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1115 ◽  
Author(s):  
Peng Jiang ◽  
Yixin Wang ◽  
Chao Liu ◽  
Yi-Chung Hu ◽  
Jingci Xie
Keyword(s):  
Decision Making ◽  
Distribution System ◽  
Multiple Attribute Decision Making ◽  
Supply System ◽  
Critical Factors ◽  
Logistics System ◽  
Factors Influencing ◽  
Emergency Logistics ◽  
Multiple Attribute ◽  
Coordination System

The infectious disease COVID-19 has swept across the world in 2020, and it continues to cause massive losses of life and severe economic problems in all countries. Providing emergency supplies such as protective medical equipment and materials required to secure people’s livelihood is thus currently prioritized by governments. Establishing a reliable emergency logistics system is critical in this regard. This paper used the Delphi method to design a formal decision structure to assess emergency logistics system reliability (ELSR) by obtaining a consensus from a panel of experts. Assessing ELSR is a typical multiple-attribute decision making (MADM) problem, and the related MADM methods are usually on the basis of symmetry principles. A hybrid MADM model, called the Decision Making Trial and Evaluation Laboratory (DEMATEL)-based Analytical Network Process (D-ANP), was developed to identify the critical factors influencing ELSR. An analysis of empirical evidence showed that the emergency logistics command and coordination system and the emergency material supply system play important roles in ELSR, while the emergency logistics transportation and distribution system and the emergency information system are not so important. This conclusion is different from previous research about traditional disaster emergency logistics. Moreover, the cause–effect relationships among the key factors indicated that the system of command and coordination for emergency logistics and the supply system for emergency materials should be improved. Accordingly, effective suggestions for emergency logistics services for epidemic prevention are provided in this paper. The main contributions of this paper are (1) establishing a comprehensive and systematic evaluating index of ELSR for epidemic prevention; (2) employing a kind of structured, namely D-ANP, to identify the critical factors with non-commensurable and conflicting (competing) characteristics; and (3) comparing the differences of reliable criteria between the emergency logistics of epidemic prevention and the traditional disaster emergency logistics.

scholarly journals Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomoko Nakanishi ◽  
Aya Maekawa ◽  
Mariko Suzuki ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
...  
Keyword(s):  
Target Gene ◽  
Animal Experiments ◽  
Adenovirus Vector ◽  
Mouse Embryonic Fibroblast ◽  
Newborn Mouse ◽  
Guide Rnas ◽  
One Step ◽  
Target Effect

AbstractSimultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

CRITICAL FACTORS INFLUENCING IMPLEMENTATION OF ROAD PROJECTS IN KENYA

2019 ◽  
Vol 4 (2) ◽  
pp. 1-22
Author(s):  
Nelly Jebitok ◽  
DR. Joyce Nzulwa
Keyword(s):  
Project Management ◽  
Survey Design ◽  
Target Population ◽  
Coefficient Of Determination ◽  
Critical Factors ◽  
Policy And Practice ◽  
Water Projects ◽  
Factors Influencing ◽  
The Government ◽  
Resource Support

Purpose: The Purpose of the study was to establish Critical factors influencing implementation of road projects.Methodology: The study adopted a descriptive survey design. The target population of the study was all the road engineers, middle managers in department of KRB. The sample size was 188 respondents. Data collected was cleaned, pretested, validated, and coded, summarized and analyzed using statistical package of SPSS V23.  The study findings were presented using graphs, histograms, bar charts and pie charts.  Conclusions were derived based on the P.value and the coefficient of determination.  Results: The study found that the key significant determinants of sustainability of water projects in Machakos County were capacity of the project management, government policies, monitoring and resource support. The study concluded that project management capacity had the greatest determinant ofsustainabilityofwater projects in Machakos County, followed by resource support, then monitoring while government policy had the least determinant of sustainability of water projects in Machakos County.Contribution to policy and practice: The study recommends that the government should advocate for proper planning with involvement of the benefiting community and timely implementation with the required results. This can be done through making of a policy by the ministry demanding for the practice of the same by the involved organizations. The project committee should set up financial structures considering both rising of funds and dissemination of the same in relation to operating and maintaining of the project. This can be done through learning and training on the same. The study also recommended that water beneficiaries and management should be sensitized to improve their knowledge on conservation and protection of water facilities.

scholarly journals Factors Influencing on E-Banking Practices: Evidence from Sri Lanka

2017 ◽  
Vol 9 (1) ◽  
pp. 1 ◽  
Author(s):  
Anusiga Gunaratnam ◽  
Konalingam Kajenthiran ◽  
Umanakenan Ratnam ◽  
Achchuthan Sivapalan
Keyword(s):  
Factor Analysis ◽  
Sri Lanka ◽  
Influencing Factors ◽  
Likert Scale ◽  
Total Variance ◽  
Critical Factors ◽  
Factors Influencing ◽  
Unique Model

The purpose of this paper is to explore the influencing factors of e-banking practices in Sri Lanka. A questionnaire with seven-point Likert scale is applied to 388 usable responses. The study was exploratory in nature and employed factor analysis to identify the important factors of e-banking practices. Results indicate that mainly four factors are significant with respect to the e-banking practices. Privacy & convenience, content & website layout, speed of delivery, and accessibility are the critical factors influencing the e-banking practices. In addition, factors extracted from the analysis accounted for 53.704% of the total variance. This study expected to provide a unique model in the realm of e-banking.

scholarly journals Critical Factors Influencing CO2 Capture Cost, a Case Study

2014 ◽  
Vol 63 ◽  
pp. 7298-7306 ◽  
Author(s):  
Ragnhild Skagestad ◽  
Agnieszka Lach ◽  
Nils Røkke ◽  
Nils Henrik Eldrup
Keyword(s):  
Co2 Capture ◽  
Critical Factors ◽  
Factors Influencing

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 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.

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