Applications of genome editing by programmable nucleases to the metabolic engineering of secondary metabolites

2017 ◽  
Vol 241 ◽  
pp. 50-60 ◽  
Author(s):  
Ana Lúcia Leitão ◽  
Marina C. Costa ◽  
Francisco J. Enguita
Keyword(s):  
Metabolic Engineering ◽  
Secondary Metabolites ◽  
Genome Editing ◽  
Programmable Nucleases

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 Target-AID-Mediated Multiplex Base Editing in Porcine Fibroblasts

2021 ◽  
Author(s):  
Soo-Young Yum ◽  
Goo Jang ◽  
Okjae Koo
Keyword(s):  
Genome Editing ◽  
Cytidine Deaminase ◽  
Chromosomal Rearrangements ◽  
Dna Breaks ◽  
Base Editing ◽  
Multiple Loci ◽  
Double Strand Dna Breaks ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Programmable Nucleases

Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3-5 base editing windows, 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

scholarly journals Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Vijaydev Ganesan ◽  
Michael Spagnuolo ◽  
Ayushi Agrawal ◽  
Spencer Smith ◽  
Difeng Gao ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Yarrowia Lipolytica ◽  
Gene Editing ◽  
Molecular Genetic ◽  
Industrial Applications ◽  
Cell Factory ◽  
Fuel Feed ◽  
Renewable Chemicals ◽  
Pharmaceutical Applications

AbstractYarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR–Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y. lipolytica. In this review we summarize the work to develop these tools and their demonstrated uses in engineering Y. lipolytica, discuss important subtleties and challenges to using these tools, and give our perspective on important gaps in gene/genome editing tools in Y. lipolytica.

Therapeutic genome editing with engineered nucleases

2017 ◽  
Vol 37 (01) ◽  
pp. 45-52 ◽  
Author(s):  
Simone Haas ◽  
Viviane Dettmer ◽  
Toni Cathomen
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Genetic Disorders ◽  
Zinc Finger Nucleases ◽  
New Era ◽  
Engineered Nucleases ◽  
Genetic Interventions ◽  
Type Gene ◽  
Programmable Nucleases ◽  
Cancer Immunotherapies

SummaryTargeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies. Here, we review the development of the different classes of programmable nucleases, discuss the challenges and improvements in translating gene editing into clinical use, and give an outlook on what applications can expect to enter the clinic in the near future.

scholarly journals Biochemically diverse CRISPR-Cas9 orthologs

2020 ◽  
Author(s):  
Giedrius Gasiunas ◽  
Joshua K. Young ◽  
Tautvydas Karvelis ◽  
Darius Kazlauskas ◽  
Tomas Urbaitis ◽  
...  
Keyword(s):  
Temperature Dependence ◽  
Genome Editing ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Programmable Nucleases ◽  
Computational Analyses ◽  
Sequence Requirements ◽  
Insight Into ◽  
Biochemical Diversity

ABSTRACTCRISPR-Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity, we applied cell-free biochemical screens to rapidly assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of novel Cas9 proteins. This approach permitted the characterization of 79 Cas9 orthologs with at least 7 distinct classes of gRNAs and 50 different PAM sequence requirements. PAM recognition spanned the entire spectrum of T-, A-, C-, and G-rich nucleotides ranging from simple di-nucleotide recognition to complex sequence strings longer than 4. Computational analyses indicated that most of this diversity came from 4 groups of interrelated sequences providing new insight into Cas9 evolution and efforts to engineer PAM recognition. A subset of Cas9 orthologs were purified and their activities examined further exposing additional biochemical diversity. This constituted both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for longer stretches of homology between gRNA and DNA target to function robustly. In all, the diverse collection of Cas9 orthologs presented here sheds light on Cas9 evolution and provides a rich source of PAM recognition and other potentially desirable properties that may be mined to expand the genome editing toolbox with new RNA-programmable nucleases.

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.

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