scholarly journals Discovering functional sequences with RELICS, an analysis method for tiling CRISPR screens

2019 ◽  
Author(s):  
Patrick C. Fiaux ◽  
Hsiuyi V. Chen ◽  
Aaron R. Chen ◽  
Poshen B. Chen ◽  
Graham McVicker
Keyword(s):  
New Technology ◽  
Regulatory Sequences ◽  
Analysis Method ◽  
Genome Sequences ◽  
Bayesian Hierarchical ◽  
Guide Rna ◽  
Target Sites ◽  
Cellular Phenotypes ◽  
Genomic Regions ◽  
Crispr Activation

AbstractCRISPR screens are a powerful new technology for the identification of genome sequences that affect cellular phenotypes such as gene expression, survival, and proliferation. By tiling single-guide RNA (sgRNA) target sites across large genomic regions, CRISPR screens have the potential to systematically discovery novel functional sequences, however, a lack of purpose-built analysis tools limits the effectiveness of this approach. Here we describe RELICS, a Bayesian hierarchical model for the discovery of functional sequences from tiling CRISPR screens. RELICS considers the overlapping effects of multiple nearby functional sequences, accounts for the ‘area of effect’ surrounding sgRNA target sites, models overdispersion in sgRNA counts, combines information across multiple pools, and estimates the number of functional sequences supported by the data. In simulations, RELICS outperforms existing methods and provides higher resolution predictions. We apply RELICS to published CRISPR interference and CRISPR activation screens and predict novel regulatory sequences, several of which we experimentally validate. In summary, RELICS is a powerful new analysis method for tiling CRISPR screens that enables the discovery of functional sequences with unprecedented resolution and accuracy.

Blockchain in Supply Chain: Case Study of Lottemart Ciputat

2019 ◽  
Vol 5 (1) ◽  
pp. 38-49 ◽  
Author(s):  
B. K. Handoyo ◽  
M. R. Mashudi ◽  
H. P. Ipung
Keyword(s):  
Decision Making ◽  
Supply Chain ◽  
Supply Chain Management ◽  
Information Asymmetry ◽  
New Technology ◽  
Business Case ◽  
Analysis Method ◽  
Chain Management ◽  
Production Processes

Current supply chain methods are having difficulties in resolving problems arising from the lack of trust in supply chains. The root reason lies in two challenges brought to the traditional mechanism: self-interests of supply chain members and information asymmetry in production processes. Blockchain is a promising technology to address these problems. The key objective of this paper is to present qualitative analysis for blockchain in supply chain as the decision-making framework to implement this new technology. The analysis method used Val IT business case framework, validated by the expert judgements. The further study needs to be elaborated by either the existing organization that use blockchain or assessment by the organization that will use blockchain to improve their supply chain management.

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.

scholarly journals Human genomic regions with exceptionally high levels of population differentiation identified from 911 whole-genome sequences

2014 ◽  
Vol 15 (6) ◽  
pp. R88 ◽  
Author(s):  
Vincenza Colonna ◽  
Qasim Ayub ◽  
Yuan Chen ◽  
Luca Pagani ◽  
Pierre Luisi ◽  
...  
Keyword(s):  
Population Differentiation ◽  
Whole Genome ◽  
Genome Sequences ◽  
Human Genomic ◽  
Genomic Regions

scholarly journals Identification of SPPL2a Inhibitors by Multiparametric Analysis of a High-Content Ultra-High-Throughput Screen

2017 ◽  
Vol 22 (9) ◽  
pp. 1106-1119 ◽  
Author(s):  
Xian Zhang ◽  
Marjo Götte ◽  
Yvonne Ibig-Rehm ◽  
Ansgar Schuffenhauer ◽  
Marion Kamke ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Trafficking ◽  
Drug Target ◽  
Nuclear Translocation ◽  
State Of The Art ◽  
Screening Strategy ◽  
Analysis Method ◽  
Intracellular Protein ◽  
Multiparametric Analysis ◽  
Cellular Phenotypes

The intramembrane protease signal peptide peptidase-like 2a (SPPL2a) is a potential drug target for the treatment of autoimmune diseases due to an essential role in B cells and dendritic cells. To screen a library of 1.4 million compounds for inhibitors of SPPL2a, we developed an imaging assay detecting nuclear translocation of the proteolytically released cytosolic substrate fragment. The state-of-the-art hit calling approach based on nuclear translocation resulted in numerous false-positive hits, mainly interrupting intracellular protein trafficking. To filter the false positives, we extracted 340 image-based readouts and developed a novel multiparametric analysis method that successfully triaged the primary hit list. The identified scaffolds were validated by demonstrating activity on endogenous SPPL2a and substrate CD74/p8 in B cells. The multiparametric analysis discovered diverse cellular phenotypes and provided profiles for the whole library. The principle of the presented imaging assay, the screening strategy, and multiparametric analysis are potentially applicable in future screening campaigns.

On the organisation of the regulatory region of the zebrafish deltaD gene

Development ◽  
2002 ◽  
Vol 129 (20) ◽  
pp. 4773-4784 ◽  
Author(s):  
Stefan Hans ◽  
José A. Campos-Ortega
Keyword(s):  
Genomic Dna ◽  
Transcriptional Control ◽  
Regulatory Region ◽  
Notch Pathway ◽  
Notch Receptor ◽  
Regulatory Sequences ◽  
Early Neurogenesis ◽  
Regulatory Feedback Loop ◽  
E Boxes ◽  
Genomic Regions

deltaD is one of the four zebrafish Delta homologues presently known. Experimental evidence indicates that deltaD participates in a number of important processes during embryogenesis, including early neurogenesis and somitogenesis, whereby the protein it encodes acts as a ligand for members of the Notch receptor family. In accordance with its functional role, deltaD is transcribed in several domains of mesodermal and ectodermal origin during embryogenesis. We have analysed the organisation of the regulatory region of the deltaD gene using fusions to the reporter gene gfp and germline transgenesis. Cis-regulatory sequences are dispersed over a stretch of 12.5 kb of genomic DNA, and are organised in a similar manner to those in the regulatory region of the Delta-like 1 gene of mouse. Germline transformation using a minigene comprising 10.5 kb of this genomic DNA attached to the 3′ end of a full-length cDNA clone rescues the phenotype of embryos homozygous for the amorphic deltaD mutation after eightAR33. Several genomic regions that drive transcription in mesodermal and neuroectodermal domains have been identified. Transcription in all the neural expression domains, with one exception, is controlled by two relatively small genomic regions, which are regulated by the proneural proteins neurogenin 1 and zash1a/b acting as transcriptional activators that bind to so-called E-boxes. Transcriptional control of deltaD by proneural proteins therefore represents a molecular target for the regulatory feedback loop mediated by the Notch pathway in lateral inhibition.

scholarly journals Patterns of Genomic Sequence Diversity among Their Simian Immunodeficiency Viruses Suggest that L'Hoest Monkeys (Cercopithecus lhoesti) Are a Natural Lentivirus Reservoir

2000 ◽  
Vol 74 (8) ◽  
pp. 3892-3898 ◽  
Author(s):  
Brigitte E. Beer ◽  
Elizabeth Bailes ◽  
George Dapolito ◽  
Barbara J. Campbell ◽  
Robert M. Goeken ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Democratic Republic ◽  
Genomic Sequence ◽  
Democratic Republic Of Congo ◽  
Vervet Monkeys ◽  
Genome Sequences ◽  
Distinct Cluster ◽  
Immunodeficiency Virus ◽  
Genomic Regions ◽  
Full Length Genome

ABSTRACT Recently, we described a novel simian immunodeficiency virus (SIVlhoest) from a wild-caught L'Hoest monkey (Cercopithecus lhoesti) from a North American zoo. To investigate whether L'Hoest monkeys are the natural host for these viruses, we have screened blood samples from 14 wild animals from the Democratic Republic of Congo. Eight (57%) were found to be seropositive for SIV. Nearly full-length genome sequences were obtained for SIV isolates from three of these monkeys and compared to the original isolate and to other SIVs. The four samples of SIVlhoest formed a distinct cluster in phylogenetic trees. Two of these isolates differed on average at only about 5% of nucleotides, suggesting that they were epidemiologically linked; otherwise, the SIVlhoest isolates differed on average by 18%. Both the level of diversity and the pattern of its variation along the genome were very similar to those seen among isolates of SIVagm from vervet monkeys, pointing to similarities in the nature of, and constraints on, SIV evolution in these two species. Discordant phylogenetic relationships among the SIVlhoest isolates for different genomic regions indicated that mosaic viruses have been generated by recombination, implying that individual monkeys have been coinfected by more than one strain of SIV. Taken together, these observations provide strong evidence that L'Hoest monkeys constitute a natural reservoir for SIV.

scholarly journals Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Ida Höijer ◽  
Josefin Johansson ◽  
Sanna Gudmundsson ◽  
Chen-Shan Chin ◽  
Ignas Bunikis ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Human Fibroblasts ◽  
Target Prediction ◽  
Human Cell Line ◽  
Cleavage Sites ◽  
Guide Rna ◽  
Target Sites ◽  
Long Read ◽  
Target Activity

Abstract Background One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro. Results The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites. Conclusions Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

scholarly journals Complete genomes for hepatitis C virus subtypes 6f, 6i, 6j and 6m: viral genetic diversity among Thai blood donors and infected spouses

2007 ◽  
Vol 88 (5) ◽  
pp. 1505-1518 ◽  
Author(s):  
Ling Lu ◽  
Chunhua Li ◽  
Yongshui Fu ◽  
Lakkana Thaikruea ◽  
Satawat Thongswat ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Complete Genome ◽  
Blood Donors ◽  
Pairwise Comparison ◽  
Genome Sequences ◽  
New Information ◽  
Hcv Transmission ◽  
Similar Range ◽  
Genomic Regions

In this study, the first complete genome sequences for hepatitis C virus (HCV) subtypes 6f, 6i, 6j and 6m, obtained from infected blood donors in Chiang Mai, Thailand, are reported. Pairwise genome-wide nucleotide similarities between some of these isolates were higher than the 75–80 % value used previously to define different HCV subtypes. To investigate further, the entire genomes of four prototype isolates, Th602 (6i), Th553 (6j), B4/92 (6m) and D86/93 (6n), were sequenced. Pairwise comparison of these sequences gave a similar range of nucleotide similarities, thereby providing new information for HCV subtype classification. In order to study the hypothesis of interspousal HCV transmission, four additional complete HCV genome sequences were obtained from two infected Thai blood donors and their spouses, C-0044 and C-0046 (6f), and C-0192 and C-0185 (6m). Pairwise comparison of the sequences revealed that C-0044 and C-0046 share a nucleotide similarity of 98.1 %, whilst C-0185 and C-0192 have a similarity of 97.8 %. Several other studies of partial HCV sequences of different genomic regions from HCV-infected couples have shown nucleotide similarities ranging from 96.3 to 100 %. The similarities of the complete genome sequences from the two couples in the current study are consistent with HCV transmission between spouses.

scholarly journals Design and Generation of MLPA Probe Sets for Combined Copy Number and Small-Mutation Analysis of Human Genes: EGFR as an Example

2010 ◽  
Vol 10 ◽  
pp. 2003-2018 ◽  
Author(s):  
Malgorzata Marcinkowska ◽  
Kwok-Kin Wong ◽  
David J. Kwiatkowski ◽  
Piotr Kozlowski
Keyword(s):  
Mutation Analysis ◽  
Copy Number ◽  
Analysis Method ◽  
Copy Number Analysis ◽  
Short Oligonucleotide ◽  
Human Genes ◽  
Simple Protocol ◽  
Commercial Kits ◽  
Genomic Regions ◽  
Dependent Probe

Multiplex ligation-dependent probe amplification (MLPA) is a multiplex copy number analysis method that is routinely used to identify large mutations in many clinical and research labs. One of the most important drawbacks of the standard MLPA setup is a complicated, and therefore expensive, procedure of generating long MLPA probes. This drawback substantially limits the applicability of MLPA to those genomic regions for which ready-to-use commercial kits are available. Here we present a simple protocol for designing MLPA probe sets that are composed entirely of short oligonucleotide half-probes generated through chemical synthesis. As an example, we present the design and generation of an MLPA assay for parallel copy number and small-mutation analysis of theEGFRgene.

scholarly journals Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci

2017 ◽  
Vol 114 (52) ◽  
pp. E11257-E11266 ◽  
Author(s):  
Samuel Lessard ◽  
Laurent Francioli ◽  
Jessica Alfoldi ◽  
Jean-Claude Tardif ◽  
Patrick T. Ellinor ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Treatment Failure ◽  
Genome Editing ◽  
Wide Spectrum ◽  
Adverse Outcomes ◽  
Human Genetic Variation ◽  
Nucleotide Polymorphisms ◽  
Guide Rna ◽  
Cas9 Nuclease ◽  
Target Sites

The CRISPR-Cas9 nuclease system holds enormous potential for therapeutic genome editing of a wide spectrum of diseases. Large efforts have been made to further understanding of on- and off-target activity to assist the design of CRISPR-based therapies with optimized efficacy and safety. However, current efforts have largely focused on the reference genome or the genome of cell lines to evaluate guide RNA (gRNA) efficiency, safety, and toxicity. Here, we examine the effect of human genetic variation on both on- and off-target specificity. Specifically, we utilize 7,444 whole-genome sequences to examine the effect of variants on the targeting specificity of ∼3,000 gRNAs across 30 therapeutically implicated loci. We demonstrate that human genetic variation can alter the off-target landscape genome-wide including creating and destroying protospacer adjacent motifs (PAMs). Furthermore, single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) can result in altered on-target sites and novel potent off-target sites, which can predispose patients to treatment failure and adverse effects, respectively; however, these events are rare. Taken together, these data highlight the importance of considering individual genomes for therapeutic genome-editing applications for the design and evaluation of CRISPR-based therapies to minimize risk of treatment failure and/or adverse outcomes.

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