scholarly journals Enhanced guide-RNA Design and Targeting Analysis for Precise CRISPR Genome Editing of Single and Consortia of Industrially Relevant and Non-Model Organisms

2017 ◽  
Author(s):  
Brian J. Mendoza ◽  
Cong T. Trinh
Keyword(s):  
Metabolic Engineering ◽  
Synthetic Biology ◽  
Genome Editing ◽  
Population Analysis ◽  
Strain Engineering ◽  
Pathway Engineering ◽  
Model Organisms ◽  
Guide Rna ◽  
Novel Applications ◽  
Rna Design

AbstractMotivationGenetic diversity of non-model organisms offers a repertoire of unique phenotypic features for exploration and cultivation for synthetic biology and metabolic engineering applications. To realize this enormous potential, it is critical to have an efficient genome editing tool for rapid strain engineering of these organisms to perform novel programmed functions.ResultsTo accommodate the use of CRISPR/Cas systems for genome editing across organisms, we have developed a novel method, named CASPER (CRISPR Associated Software for Pathway Engineering and Research), for identifying on- and off-targets with enhanced predictability coupled with an analysis of non-unique (repeated) targets to assist in editing any organism with various endonucleases. Utilizing CASPER, we demonstrated a modest 2.4% and significant 30.2% improvement (F-test, p<0.05) over the conventional methods for predicting on- and off-target activities, respectively. Further we used CASPER to develop novel applications in genome editing: multitargeting analysis (i.e. simultaneous multiple-site modification on a target genome with a sole guide-RNA (gRNA) requirement) and multispecies population analysis (i.e. gRNA design for genome editing across a consortium of organisms). Our analysis on a selection of industrially relevant organisms revealed a number of non-unique target sites associated with genes and transposable elements that can be used as potential sites for multitargeting. The analysis also identified shared and unshared targets that enable genome editing of single or multiple genomes in a consortium of interest. We envision CASPER as a useful platform to enhance the precise CRISPR genome editing for metabolic engineering and synthetic biology applications.

scholarly journals Constraint-based metabolic control analysis for rational strain engineering

2020 ◽  
Author(s):  
Sophia Tsouka ◽  
Meric Ataman ◽  
Tuure Hameri ◽  
Ljubisa Miskovic ◽  
Vassily Hatzimanikatis
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Metabolic Control ◽  
Metabolic Flux ◽  
Strain Engineering ◽  
Metabolic Control Analysis ◽  
Physiological Data ◽  
Response Analysis ◽  
Control Analysis ◽  
Wide Range

AbstractThe advancements in genome editing techniques over the past years have rekindled interest in rational metabolic engineering strategies. While Metabolic Control Analysis (MCA) is a well-established method for quantifying the effects of metabolic engineering interventions on flows in metabolic networks and metabolic concentrations, it fails to account for the physiological limitations of the cellular environment and metabolic engineering design constraints. We report here a constraint-based framework based on MCA, Network Response Analysis (NRA), for the rational genetic strain design that incorporates biologically relevant constraints, as well as genome editing restrictions. The NRA core constraints being similar to the ones of Flux Balance Analysis, allow it to be used for a wide range of optimization criteria and with various physiological constraints. We show how the parametrization and introduction of biological constraints enhance the NRA formulation compared to the classical MCA approach, and we demonstrate its features and its ability to generate multiple alternative optimal strategies given several user-defined boundaries and objectives. In summary, NRA is a sophisticated alternative to classical MCA for rational metabolic engineering that accommodates the incorporation of physiological data at metabolic flux, metabolite concentration, and enzyme expression levels.

scholarly journals SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes

DNA Research ◽  
2019 ◽  
Vol 26 (4) ◽  
pp. 327-339 ◽  
Author(s):  
Ariadna Montero-Blay ◽  
Samuel Miravet-Verde ◽  
Maria Lluch-Senar ◽  
Carlos Piñero-Lambea ◽  
Luis Serrano
Keyword(s):  
Antibiotic Resistance ◽  
Synthetic Biology ◽  
Genome Editing ◽  
Mycoplasma Pneumoniae ◽  
Mycoplasma Gallisepticum ◽  
Model Organisms ◽  
Mycoplasma Species ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker ◽  
Reference Tool

Abstract Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae, Mycoplasma feriruminatoris, Mycoplasma gallisepticum and Mycoplasma pneumoniae, selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum, M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae, basing our study on more than 199,000 genome insertions.

Guide RNA Design for CRISPR/Cas9-Mediated Potato Genome Editing

2018 ◽  
Vol 479 (1) ◽  
pp. 90-94 ◽  
Author(s):  
A. V. Khromov ◽  
V. A. Gushchin ◽  
V. I. Timerbaev ◽  
N. O. Kalinina ◽  
M. E. Taliansky ◽  
...  
Keyword(s):  
Genome Editing ◽  
Potato Genome ◽  
Guide Rna ◽  
Rna Design

scholarly journals kRISP-meR: A Reference-free Guide-RNA Design Tool for CRISPR/Cas9

2019 ◽  
Author(s):  
Mahmudur Rahman Hera ◽  
Amatur Rahman ◽  
Atif Rahman
Keyword(s):  
Genome Editing ◽  
Design Tool ◽  
Target Region ◽  
Guide Rna ◽  
Guide Rnas ◽  
Rna Design ◽  
Reference Genomes ◽  
Target Effects

AbstractGenome editing using the CRISPR/Cas9 system requires designing guide RNAs (sgRNA) that are efficient and specific. Guide RNAs are usually designed using reference genomes which limits their use in organisms with no or incomplete reference genomes. Here, we present kRISP-meR, a reference free method to design sgRNAs for CRISPR/Cas9 system. kRISP-meR takes as input a target region and sequenced reads from the organism to be edited and generates sgRNAs that are likely to minimize off-target effects. Our analysis indicates that kRISP-meR is able to identify majority of the guides identified by a widely used sgRNA designing tool, without any knowledge of the reference, while retaining specificity.

scholarly journals SNP-CRISPR: a web tool for SNP-specific genome editing

2019 ◽  
Author(s):  
Chiao-Lin Chen ◽  
Jonathan Rodiger ◽  
Verena Chung ◽  
Raghuvir Viswanatha ◽  
Stephanie E. Mohr ◽  
...  
Keyword(s):  
Genome Editing ◽  
Model Systems ◽  
Model Organisms ◽  
Data Sets ◽  
Nucleotide Polymorphisms ◽  
Web Tool ◽  
Guide Rna ◽  
Wide Range ◽  
Sgrna Design ◽  
Reference Genomes

ABSTRACTCRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction.

scholarly journals Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

2020 ◽  
Author(s):  
Xinyi Guo ◽  
Hans-Hermann Wessels ◽  
Alejandro Méndez-Mancilla ◽  
Daniel Haro ◽  
Neville E. Sanjana
Keyword(s):  
Rna Virus ◽  
H1n1 Influenza ◽  
Model Organisms ◽  
Messenger Rnas ◽  
Protein Coding ◽  
Guide Rna ◽  
Knock Down ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Rna Design

AbstractCRISPR-Cas13 mediates robust transcript knockdown in human cells through direct RNA targeting. Compared to DNA-targeting CRISPR enzymes like Cas9, RNA targeting by Cas13 is transcript- and strand-specific: It can distinguish and specifically knock-down processed transcripts, alternatively spliced isoforms and overlapping genes, all of which frequently serve different functions. Previously, we identified optimal design rules for RfxCas13d guide RNAs (gRNAs), and developed a computational model to predict gRNA efficacy for all human protein-coding genes. However, there is a growing interest to target other types of transcripts, such as noncoding RNAs (ncRNAs) or viral RNAs, and to target transcripts in other commonly-used organisms. Here, we predicted relative Cas13-driven knock-down for gRNAs targeting messenger RNAs and ncRNAs in six model organisms (human, mouse, zebrafish, fly, nematode and flowering plants) and four abundant RNA virus families (SARS-CoV-2, HIV-1, H1N1 influenza and MERS). To allow for more flexible gRNA efficacy prediction, we also developed a web-based application to predict optimal gRNAs for any RNA target entered by the user. Given the lack of Cas13 guide design tools, we anticipate this resource will facilitate CRISPR-Cas13 RNA targeting in common model organisms, emerging viral threats to human health, and novel RNA targets.

scholarly journals SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing

2019 ◽  
Vol 10 (2) ◽  
pp. 489-494 ◽  
Author(s):  
Chiao-Lin Chen ◽  
Jonathan Rodiger ◽  
Verena Chung ◽  
Raghuvir Viswanatha ◽  
Stephanie E. Mohr ◽  
...  
Keyword(s):  
Genome Editing ◽  
Model Systems ◽  
Model Organisms ◽  
Data Sets ◽  
Nucleotide Polymorphisms ◽  
Web Tool ◽  
Guide Rna ◽  
Wide Range ◽  
Sgrna Design ◽  
Reference Genomes

CRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction.

scholarly journals A toolset of constitutive promoters for metabolic engineering ofRhodosporidium toruloides

2019 ◽  
Author(s):  
Luísa Czamanski Nora ◽  
Maren Wehrs ◽  
Joonhoon Kim ◽  
Jan-Fang Cheng ◽  
Angela Tarver ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Genetic Engineering ◽  
Strain Engineering ◽  
Pathway Engineering ◽  
Transcriptional Analysis ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Promoter Sequences ◽  
Engineering Studies ◽  
Over Time

ABSTRACTBackgroundRhodosporidium toruloidesis a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering inR. toruloides.ResultsOur data describes a set of nativeR. toruloidespromoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. We measured promoter expression strength by flow cytometry using a dual fluorescent reporter system. From these analyses, we found a total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) of potential value for genetic engineering ofR. toruloides.ConclusionsWe present a list of robust and constitutive, native promoters to facilitate genetic engineering ofR. toruloides.This set of thoroughly characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.

Sign in / Sign up

Export Citation Format

Share Document