scholarly journals Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants

2016 ◽  
Vol 170 (4) ◽  
pp. 1917-1928 ◽  
Author(s):  
Noel J. Sauer ◽  
Javier Narváez-Vásquez ◽  
Jerry Mozoruk ◽  
Ryan B. Miller ◽  
Zachary J. Warburg ◽  
...  
Keyword(s):  
Genome Editing ◽  
Engineered Nucleases ◽  
And Function

Adenoviral vector DNA for accurate genome editing with engineered nucleases

2014 ◽  
Vol 11 (10) ◽  
pp. 1051-1057 ◽  
Author(s):  
Maarten Holkers ◽  
Ignazio Maggio ◽  
Sara F D Henriques ◽  
Josephine M Janssen ◽  
Toni Cathomen ◽  
...  
Keyword(s):  
Genome Editing ◽  
Adenoviral Vector ◽  
Engineered Nucleases ◽  
Vector Dna

scholarly journals Genome Editing with Engineered Nucleases in Plants

2014 ◽  
Vol 56 (3) ◽  
pp. 389-400 ◽  
Author(s):  
Y. Osakabe ◽  
K. Osakabe
Keyword(s):  
Genome Editing ◽  
Engineered Nucleases

scholarly journals CRISPR/Cas9 Genome Editing Technology: A Valuable Tool for Understanding Plant Cell Wall Biosynthesis and Function

2020 ◽  
Vol 11 ◽  
Author(s):  
Yuan Zhang ◽  
Allan M. Showalter
Keyword(s):  
Cell Wall ◽  
Genome Editing ◽  
Gene Targeting ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cell Structure ◽  
Higher Order ◽  
Knock Out ◽  
Genetic Crossing ◽  
And Function

For the past 5 years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has appeared in the molecular biology research spotlight. As a game-changing player in genome editing, CRISPR/Cas9 technology has revolutionized animal research, including medical research and human gene therapy as well as plant science research, particularly for crop improvement. One of the most common applications of CRISPR/Cas9 is to generate genetic knock-out mutants. Recently, several multiplex genome editing approaches utilizing CRISPR/Cas9 were developed and applied in various aspects of plant research. Here we summarize these approaches as they relate to plants, particularly with respect to understanding the biosynthesis and function of the plant cell wall. The plant cell wall is a polysaccharide-rich cell structure that is vital to plant cell formation, growth, and development. Humans are heavily dependent on the byproducts of the plant cell wall such as shelter, food, clothes, and fuel. Genes involved in the assembly of the plant cell wall are often highly redundant. To identify these redundant genes, higher-order knock-out mutants need to be generated, which is conventionally done by genetic crossing. Compared with genetic crossing, CRISPR/Cas9 multi-gene targeting can greatly shorten the process of higher-order mutant generation and screening, which is especially useful to characterize cell wall related genes in plant species that require longer growth time. Moreover, CRISPR/Cas9 makes it possible to knock out genes when null T-DNA mutants are not available or are genetically linked. Because of these advantages, CRISPR/Cas9 is becoming an ideal and indispensable tool to perform functional studies in plant cell wall research. In this review, we provide perspectives on how to design CRISPR/Cas9 to achieve efficient gene editing and multi-gene targeting in plants. We also discuss the recent development of the virus-based CRISPR/Cas9 system and the application of CRISPR/Cas9 to knock in genes. Lastly, we summarized current progress on using CRISPR/Cas9 for the characterization of plant cell wall-related genes.

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 CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice

2016 ◽  
Vol 24 (3) ◽  
pp. 564-569 ◽  
Author(s):  
Li Xu ◽  
Ki Ho Park ◽  
Lixia Zhao ◽  
Jing Xu ◽  
Mona El Refaey ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mdx Mice ◽  
Dystrophin Expression ◽  
And Function

scholarly journals A Nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding

2020 ◽  
Author(s):  
Carl W. White ◽  
Kevin D. G. Pfleger ◽  
Stephen J. Hill
Keyword(s):  
Real Time ◽  
Genome Editing ◽  
Receptor Binding ◽  
Chemokine Receptors ◽  
Live Cells ◽  
Chemokine Cxcl12 ◽  
Membrane Bound ◽  
Low Levels ◽  
And Function ◽  
Steric Constraints

SummarySecreted chemokines are critical mediators of cellular communication that elicit intracellular signalling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. CRISPR/Cas9 genome-editing was used to tag the chemokine CXCL12 with the Nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of Nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analysed using BRET. These live cell approaches combine the sensitivity of Nanoluciferase with CRISPR/Cas9 genome-editing to detect, quantify and monitor binding of low levels of native secreted proteins in real time.

scholarly journals Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence repair outcome

2018 ◽  
Author(s):  
Eirini M Kallimasioti-Pazi ◽  
Keerthi Thelakkad Chathoth ◽  
Gillian C Taylor ◽  
Alison Meynert ◽  
Tracy Ballinger ◽  
...  
Keyword(s):  
Genome Editing ◽  
Imprinted Genes ◽  
Induced Mutations ◽  
Permeable Barrier ◽  
Homology Directed Repair ◽  
Confounding Variables ◽  
Experimental Parameters ◽  
Allele Specific ◽  
And Function ◽  
Mutagenic Dna Repair

AbstractCRISPR-Cas9 genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem. Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of Cas9-induced mutations can be attributed unequivocally to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. Surprisingly, the outcome of mutagenic DNA repair was independent of chromatin state, with similar efficiencies of homology directed repair and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint of CRISPR-Cas9 genome editing, and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.

scholarly journals 231 Structure and mechanism of CRISPR/Cas9

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 56-57
Author(s):  
David W Taylor
Keyword(s):  
Human Health ◽  
Genome Editing ◽  
Genome Engineering ◽  
Structure And Function ◽  
Recent Advances ◽  
And Function

Abstract Recent advances in genome editing using CRISPR-Cas9 and related technologies have revolutionized the ability to manipulate genes in a rapid, precise, and flexible manner. These advances have spurred an explosion of interest in the possible ways in which genome editing can improve human health. I will provide an overview of CRISPR-Cas systems, the structure and function of CRISPR-Cas9, and the repurposing of CRISPR-Cas9 for genome engineering.

scholarly journals Efficient Multi-Sites Genome Editing and Plant Regeneration via Somatic Embryogenesis in Picea glauca

2021 ◽  
Vol 12 ◽  
Author(s):  
Ying Cui ◽  
Jian Zhao ◽  
Ying Gao ◽  
Ruirui Zhao ◽  
Jinfeng Zhang ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
Genome Editing ◽  
Picea Glauca ◽  
White Spruce ◽  
Embryogenic Tissue ◽  
Ecological Value ◽  
Green Plants ◽  
A Genome ◽  
And Function

Conifers are the world's major source of timber and pulpwood and have great economic and ecological value. Currently, little research on the application of CRISPR/Cas9, the commonly used genome-editing tool in angiosperms, has been reported in coniferous species. An efficient CRISPR/Cas9 system based on somatic embryogenesis (SEis) suitable for conifers could benefit both fundamental and applied research in these species. In this study, the SpCas9 gene was optimized based on codon bias in white spruce, and a spruce U6 promoter was cloned and function-validated for use in a conifer specific CRISPR/Cas9 toolbox, i.e., PgCas9/PaU6. With this toolbox, a genome-editing vector was constructed to target the DXS1 gene of white spruce. By Agrobacterium-mediated transformation, the genome-editing vector was then transferred into embryogenic tissue of white spruce. Three resistant embryogenic tissues were obtained and used for regenerating plants via SEis. Albino somatic embryo (SE) plants with mutations in DXS1 were obtained in all of the three events, and the ratios of the homozygous and biallelic mutants in the 18 albino mutants detected were 22.2% in both cases. Green plants with mutations in DXS1 were also produced, and the ratios of the DXS1 mutants to the total green plants were 7.9, 28, and 13.5%, respectively, among the three events. Since 22.7% of the total 44 mutants were edited at both of the target sites 1 and 2, the CRISPR/Cas9 toolbox in this research could be used for multi-sites genome editing. More than 2,000 SE plants were regenerated in vitro after genome editing, and part of them showed differences in plant development. Both chimerism and mosaicism were found in the SE plants of white spruce after genome editing with the CRISPR/Cas9 toolbox. The conifer-specific CRISPR/Cas9 system developed in this research could be valuable in gene function research and trait improvement.

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