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Open accessJournal ArticleDOI: 10.1186/S12859-021-04034-6

PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants.

02 Mar 2021-BMC Bioinformatics (Springer Science and Business Media LLC)-Vol. 22, Iss: 1, pp 101-101
Abstract: The rapid expansion of the CRISPR toolbox through tagging effector domains to either enzymatically inactive Cas9 (dCas9) or Cas9 nickase (nCas9) has led to several promising new gene editing strategies. Recent additions include CRISPR cytosine or adenine base editors (CBEs and ABEs) and the CRISPR prime editors (PEs), in which a deaminase or reverse transcriptase are fused to nCas9, respectively. These tools hold great promise to model and correct disease-causing mutations in animal and plant models. But so far, no widely-available tools exist to automate the design of both BE and PE reagents. We developed PnB Designer, a web-based application for the design of pegRNAs for PEs and guide RNAs for BEs. PnB Designer makes it easy to design targeting guide RNAs for single or multiple targets on a variant or reference genome from organisms spanning multiple kingdoms. With PnB Designer, we designed pegRNAs to model all known disease causing mutations available in ClinVar. Additionally, PnB Designer can be used to design guide RNAs to install or revert a SNV, scanning the genome with one CBE and seven different ABE PAM variants and returning the best BE to use. PnB Designer is publicly accessible at http://fgcz-shiny.uzh.ch/PnBDesigner/ With PnB Designer we created a user-friendly design tool for CRISPR PE and BE reagents, which should simplify choosing editing strategy and avoiding design complications.

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Topics: Cas9 (51%)
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23 results found


Open accessJournal ArticleDOI: 10.1016/J.TPLANTS.2021.06.015
Mahmudul Hassan1, Mahmudul Hassan2, Yingxiao Zhang3, Guoliang Yuan2  +6 moreInstitutions (3)
Abstract: CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for high-throughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing.

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Topics: CRISPR (60%), Genome editing (58%)

3 Citations


Open accessPosted ContentDOI: 10.1101/2021.08.26.457758
Marina A. Pak1, Dmitry N. Ivankov1Institutions (1)
27 Aug 2021-bioRxiv
Abstract: MotivationPrediction of protein stability change upon mutation ({Delta}{Delta}G) is crucial for facilitating protein engineering and understanding of protein folding principles. Robust prediction of protein folding free energy change requires the knowledge of protein three-dimensional (3D) structure. Unfortunately, protein 3D structure is not always available. In this case, one can still predict the protein stability change by constructing a homology model of the protein; however, the accuracy of homology model-based {Delta}{Delta}G predictions is unknown. The perspectives of using 3D structures of the best templates are also unclear. ResultsTo investigate these questions, we used the most popular and accurate publicly available tools: FoldX for stability change prediction and I-Tasser for homology modeling. We found that both homology models and best templates worsen the {Delta}{Delta}G prediction, with best templates performing 1.5 times better than homology models. For AlphaFold models, we also found that the best templates seem to outperform protein models. Our findings imply using the 3D structures of the best templates for {Delta}{Delta}G prediction if the 3D protein structure is unavailable. Contactd.ivankov@skoltech.ru

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Topics: FoldX (53%), Homology modeling (51%)

2 Citations


Journal ArticleDOI: 10.1038/S41477-021-00991-1
01 Sep 2021-Nature plants
Abstract: The development of CRISPR–Cas systems has sparked a genome editing revolution in plant genetics and breeding. These sequence-specific RNA-guided nucleases can induce DNA double-stranded breaks, resulting in mutations by imprecise non-homologous end joining (NHEJ) repair or precise DNA sequence replacement by homology-directed repair (HDR). However, HDR is highly inefficient in many plant species, which has greatly limited precise genome editing in plants. To fill the vital gap in precision editing, base editing and prime editing technologies have recently been developed and demonstrated in numerous plant species. These technologies, which are mainly based on Cas9 nickases, can introduce precise changes into the target genome at a single-base resolution. This Review provides a timely overview of the current status of base editors and prime editors in plants, covering both technological developments and biological applications. This Review summarizes the current status of base editors and prime editors in plants, reporting both technological developments and biological applications of these precise genome editors.

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Topics: Genome editing (58%)

2 Citations


Open accessJournal ArticleDOI: 10.1016/J.JNEUMETH.2021.109373
Abstract: Background The classification of motor imagery electroencephalogram (MI-EEG) is a pivotal task in the biosignal classification process in the brain-computer interface (BCI) applications. Currently, this bio-engineering-based technology is being employed by researchers in various fields to develop cutting-edge applications. The classification of real-time MI-EEG signals is the most challenging task in these applications. The prediction performance of the existing classification methods is still limited due to the high dimensionality and dynamic behaviors of the real-time EEG data. Proposed method To enhance the classification performance of real-time BCI applications, this paper presents a new clustering-based ensemble technique called CluSem to mitigate this problem. We also develop a new brain game called CluGame using this method to evaluate the classification performance of real-time motor imagery movements. In this game, real-time EEG signal classification and prediction tabulation through animated balls are controlled via threads. By playing this game, users can control the movements of the balls via the brain signals of motor imagery movements without using any traditional input devices. Results Our results demonstrate that CluSem is able to improve the classification accuracy between 5% and 15% compared to the existing methods on our collected as well as the publicly available EEG datasets. The source codes used to implement CluSem and CluGame are publicly available at https://github.com/MdOchiuddinMiah/MI-BCI_ML .

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Topics: Motor imagery (53%), Cluster analysis (52%), Biosignal (52%) ... read more

Journal ArticleDOI: 10.1016/J.YMTHE.2021.09.002
03 Nov 2021-Molecular Therapy
Abstract: Recent advances in genome editing technologies have magnified the prospect of single-dose cures for many genetic diseases. For most genetic disorders, precise DNA correction is anticipated to best treat patients. To install desired DNA changes with high precision, our laboratory developed base editors (BEs), which can correct the four most common single-base substitutions, and prime editors, which can install any substitution, insertion, and/or deletion over a stretch of dozens of base pairs. Compared to nuclease-dependent editing approaches that involve double-strand DNA breaks (DSBs) and often result in a large percentage of uncontrolled editing outcomes, such as mixtures of insertions and deletions (indels), larger deletions, and chromosomal rearrangements, base editors and prime editors often offer greater efficiency with fewer byproducts in slowly dividing or non-dividing cells, such as those that make up most of the cells in adult animals. Both viral and non-viral in vivo delivery methods have now been used to deliver base editors and prime editors in animal models, establishing that base editors and prime editors can serve as effective agents for in vivo therapeutic genome editing in animals. This review summarizes examples of in vivo somatic cell (post-natal) base editing and prime editing and prospects for future development.

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Topics: Genome editing (55%)

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36 results found


Open accessJournal ArticleDOI: 10.1186/GB-2004-5-10-R80
15 Sep 2004-Genome Biology
Abstract: The Bioconductor project is an initiative for the collaborative creation of extensible software for computational biology and bioinformatics. The goals of the project include: fostering collaborative development and widespread use of innovative software, reducing barriers to entry into interdisciplinary scientific research, and promoting the achievement of remote reproducibility of research results. We describe details of our aims and methods, identify current challenges, compare Bioconductor to other open bioinformatics projects, and provide working examples.

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Topics: Bioconductor (65%)

11,488 Citations


Open accessJournal ArticleDOI: 10.1038/NATURE17946
Alexis C. Komor1, Yongjoo Kim2, Yongjoo Kim1, Michael S. Packer1  +5 moreInstitutions (2)
19 May 2016-Nature
Abstract: Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

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Topics: Genome editing (56%), DNA repair (55%), Point mutation (55%) ... read more

2,245 Citations


Open accessJournal ArticleDOI: 10.1038/NMETH.3252
Wolfgang Huber, Vincent J. Carey1, Robert Gentleman2, Simon Anders  +22 moreInstitutions (13)
01 Feb 2015-Nature Methods
Abstract: Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors.

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Topics: Bioconductor (71%)

2,202 Citations


Open accessJournal ArticleDOI: 10.1093/NAR/GKT1113
Abstract: ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/) provides a freely available archive of reports of relationships among medically important variants and phenotypes. ClinVar accessions submissions reporting human variation, interpretations of the relationship of that variation to human health and the evidence supporting each interpretation. The database is tightly coupled with dbSNP and dbVar, which maintain information about the location of variation on human assemblies. ClinVar is also based on the phenotypic descriptions maintained in MedGen (http://www.ncbi.nlm.nih.gov/medgen). Each ClinVar record represents the submitter, the variation and the phenotype, i.e. the unit that is assigned an accession of the format SCV000000000.0. The submitter can update the submission at any time, in which case a new version is assigned. To facilitate evaluation of the medical importance of each variant, ClinVar aggregates submissions with the same variation/phenotype combination, adds value from other NCBI databases, assigns a distinct accession of the format RCV000000000.0 and reports if there are conflicting clinical interpretations. Data in ClinVar are available in multiple formats, including html, download as XML, VCF or tab-delimited subsets. Data from ClinVar are provided as annotation tracks on genomic RefSeqs and are used in tools such as Variation Reporter (http://www.ncbi.nlm.nih.gov/variation/tools/reporter), which reports what is known about variation based on user-supplied locations.

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1,852 Citations


Open accessJournal ArticleDOI: 10.1038/NATURE24644
23 Nov 2017-Nature
Abstract: The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

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Topics: Base pair (58%), DNA (57%), Point mutation (56%) ... read more

1,475 Citations


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