scholarly journals Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka

2013 ◽  
Vol 56 (1) ◽  
pp. 98-107 ◽  
Author(s):  
Satoshi Ansai ◽  
Keiji Inohaya ◽  
Yasutoshi Yoshiura ◽  
Manfred Schartl ◽  
Norihito Uemura ◽  
...  
Keyword(s):  
Targeted Mutagenesis ◽  
Design Evaluation ◽  
Screening Methods ◽  
Transcription Activator

scholarly journals Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

Genetics ◽  
2013 ◽  
Vol 193 (3) ◽  
pp. 739-749 ◽  
Author(s):  
Satoshi Ansai ◽  
Tetsushi Sakuma ◽  
Takashi Yamamoto ◽  
Hiroyoshi Ariga ◽  
Norihito Uemura ◽  
...  
Keyword(s):  
Targeted Mutagenesis ◽  
Transcription Activator

scholarly journals Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox

2019 ◽  
Vol 20 (16) ◽  
pp. 4045 ◽  
Author(s):  
Ali Razzaq ◽  
Fozia Saleem ◽  
Mehak Kanwal ◽  
Ghulam Mustafa ◽  
Sumaira Yousaf ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Crop Improvement ◽  
Crop Plants ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
Zinc Finger Nucleases ◽  
Base Substitution ◽  
Transcription Activator ◽  
Abiotic Stressors

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

scholarly journals Application of genome editing tools in plants

2021 ◽  
Vol 19 (1) ◽  
pp. 15-40
Author(s):  
Nguyen Duc Thanh
Keyword(s):  
Genome Editing ◽  
Target Gene ◽  
Crop Improvement ◽  
Double Sequence ◽  
Targeted Mutagenesis ◽  
Nonhomologous End Joining ◽  
Transcription Activator ◽  
End Joining ◽  
Base Editing ◽  
Living Organisms

Genome editing technology is the genome modification techniques, such as targeted mutagenesis or insert/delete/replacement at specific locations in the genome of living organisms. Genome editing is based on the creation of double sequence break (DSB) in a specific location and DNA repair via nonhomologous end joining (NHEJ) or homology direct repair (HDR). The development of sequence-specific nuclease (SSN) allows precise editing of the target gene. These SSNs include: meganuclease (MN), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR-associated nuclease (Cas) including CRISPR/Cas9 (from Streptococcus pyogenes) and CRISPR/Cpf1 (from Prevoltella and Francisella1). These are the genome editing tools used to create DSBs at specific locations of the genome. Recently, the base editing (BE) and prime editing (PE) tools have been reported. This review will cover the basics of these tools and their application in genome editing in plants, especially providing the most up-to-date information on their application in crop improvement.

scholarly journals Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

2015 ◽  
Vol 81 (22) ◽  
pp. 7888-7892 ◽  
Author(s):  
Christopher J. Pappas ◽  
Mathieu Picardeau
Keyword(s):  
Genetic Manipulation ◽  
Immunoblot Analysis ◽  
Targeted Mutagenesis ◽  
Pcr Analysis ◽  
Leptospira Interrogans ◽  
Transcription Activator ◽  
Promoter Regions ◽  
Control Of Gene Expression ◽  
Content Type

ABSTRACTLeptospirosis is a zoonotic disease that affects ∼1 million people annually, with a mortality rate of >10%. Currently, there is an absence of effective genetic manipulation tools for targeted mutagenesis in pathogenic leptospires. Transcription activator-like effectors (TALEs) are a recently described group of repressors that modify transcriptional activity in prokaryotic and eukaryotic cells by directly binding to a targeted sequence within the host genome. To determine the applicability of TALEs withinLeptospiraspp., two TALE constructs were designed. First, a constitutively expressed TALE gene specific for thelacO-like region upstream ofbgaLwastransinserted in the saprophyteLeptospira biflexa(the TALEβgalstrain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALEβgalstrain. Second, to study the role of LigA and LigB in pathogenesis, a constitutively expressed TALE gene with specificity for the homologous promoter regions ofligAandligBwascisinserted into the pathogenLeptospira interrogans(TALElig). LigA and LigB expression was studied by using three independent clones: TALElig1, TALElig2, and TALElig3. Immunoblot analysis of osmotically induced TALEligclones demonstrated 2- to 9-fold reductions in the expression levels of LigA and LigB, with the highest reductions being noted for TALElig1and TALElig2, which were avirulentin vivoand nonrecoverable from animal tissues. This study reconfirms galactosidase activity in the saprophyte and suggests a role for LigA and LigB in pathogenesis. Collectively, this study demonstrates that TALEs are effective at reducing the expression of targeted genes within saprophytic and pathogenic strains ofLeptospiraspp., providing an additional genetic manipulation tool for this genus.

scholarly journals Targeted Mutagenesis of the Tomato PROCERA Gene Using Transcription Activator-Like Effector Nucleases

2014 ◽  
Vol 166 (3) ◽  
pp. 1288-1291 ◽  
Author(s):  
V. S. Lor ◽  
C. G. Starker ◽  
D. F. Voytas ◽  
D. Weiss ◽  
N. E. Olszewski
Keyword(s):  
Targeted Mutagenesis ◽  
Transcription Activator

scholarly journals Targeted modification of gene function exploiting homology-directed repair of TALEN-mediated double strand breaks in barley

2015 ◽  
Author(s):  
Nagaveni Budhagatapalli ◽  
Twan Rutten ◽  
Maia Gurushidze ◽  
Jochen Kumlehn ◽  
Goetz Hensel
Keyword(s):  
Dna Sequence ◽  
Gene Function ◽  
Genetically Engineered ◽  
Double Strand Breaks ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Transcription Activator ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Repair Template

Transcription activator-like effector nucleases (TALENs) open up new opportunities for targeted mutagenesis in eukaryotic genomes. Similar to zinc-finger nucleases, sequence-specific DNA-binding domains can be fused with effector domains like the nucleolytically active part of FokI in order to induce double strand breaks (DSBs) and thereby modify the host genome on a predefined target site via non-homologous end joining. More sophisticated applications of programmable endonucleases involve the use of a DNA repair template facilitating homology-directed repair (HDR) so as to create predefined rather than random DNA sequence modifications. The aim of this study was to demonstrate the feasibility of editing the barley genome by precisely modifying a defined target DNA sequence resulting in a predicted alteration of gene function. We usedgfp-specific TALENs along with a repair template that, via HDR, facilitates conversion ofgfpintoyfpwhich is associated with a single amino acid exchange in the gene product. As a result of co-bombardment of leaf epidermis, we detected YFP accumulation in about 3 out of 100 mutated cells. The creation of a functionalyfpgene via HDR was unambiguously confirmed by sequencing of the respective genomic site. Predictable genetic modifications comprising only a few genomic base pairs rather than entire genes are of particular practical relevance, because they might not fall under the European regulation of genetically engineered organisms. In addition to the allele conversion accomplished in planta, a readily screenable marker system is introduced that might be useful for optimization approaches in the field of genome editing.

TOSO Deficiency Exerts Impact On BAFF-R Expression, Thereby Causing Impaired B Cell Development in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3898-3898
Author(s):  
Alexandra Schulz ◽  
Yoon Jung Park ◽  
Rainer Claus ◽  
Hamid Kashkar ◽  
Jens Seeger ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Development ◽  
B Cell Development ◽  
Targeted Mutagenesis ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Transcription Activator ◽  
B Cell Lymphomas ◽  
Survival Signaling

Abstract Abstract 3898 Apoptosis resistance concomitant with aberrant upregulation of pro-survival pathways is a main pathogenic mechanism in development and maintenance of chronic lymphocytic leukemia (CLL). Our group recently identified TOSO to be significantly overexpressed in CLL compared to other B cell lymphomas or healthy B cells. Interestingly, TOSO is thought to exert pro-survival signaling, although it remains still enigmatic, how TOSO is regulated and why TOSO is expressed extremely heterogeneous on different B cells entities. Moreover, we previously detected elevated TOSO expression to be associated with progressive disease, including unmutated IgVH status of the B cell receptor (BCR). Since the BCR is a driving force in CLL, TOSO expression was investigated after BCR crosslinking and resulted in an increase of TOSO. To date, the TOSO promoter has not been described yet. Here, we firstly identified the TOSO proximal region to exert promoter activity. Moreover, in silico analysis and phylogenetic footprinting exhibited existence of transcription factor binding sites for NF-κB and BCL6. In luciferase reporter assays, including targeted mutagenesis, NF-κB was confirmed as novel inducer of TOSO expression. Whereas BCL6 binding, confirmed by ChIP and luciferase assays, was shown to exert repressing activity on the TOSO promoter. Although it can explained now how TOSO is regulated by the BCR, the reason for its distinct basal expression levels in normal B cells and other B cell malignancies still remained unclear. Our data illustrate for the first time that DNA hypomethylation of the TOSO promoter is a conspicuous characteristic in CLL patients compared to healthy donors. Indeed, the methylation status seems to play a major role, since the methylation level correlates with TOSO expression also in other B cell lymphomas. Moreover, it is indispensible to clarify the biologic significance of TOSO, particularly in the CLL relevant B cells. Therefore, we generated a B cell-specific knockout mouse model and identified impaired B cell development characterized by diminished B cell count. Gene expression analysis and flow cytometry revealed a decrease of the B-cell activating factor receptor (BAFF-R). BAFF-R ligation is known to promote B cell survival in particular via degradation of IκBα and translocation of NF-κB to the nucleus, thus activating the NF-κB pathway. Thus, BAFF-R decrease caused by TOSO depletion might lead to the detected reduction of B lymphocytes, which corresponds to the previous observations. Taken together, this work reveals a counteractive TOSO regulation by transcription activator NF-κB and transcription repressor BCL6 in a BCR-dependent manner in B cells. Moreover, we detected CLL-specific hypomethylation of the TOSO promoter, which is supposed to be causative for elevated TOSO level in CLL. Moreover, our results might reveal a new function of TOSO in pro-survival signaling and B cell homeostasis, supporting the anti-apoptotic feature of TOSO in B cells. Identifying the regulating mechanisms and biological function of the anti-apoptotic TOSO, is an essential step towards elucidation of the underlying molecular causes for the development of CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Raviraj Banakar ◽  
Alan L. Eggenberger ◽  
Keunsub Lee ◽  
David A. Wright ◽  
Karthik Murugan ◽  
...  
Keyword(s):  
High Frequency ◽  
Genome Engineering ◽  
Selectable Marker ◽  
Marker Gene ◽  
Targeted Mutagenesis ◽  
Screening Methods ◽  
Chromosomal Dna ◽  
Target Sites ◽  
Rnp Complex ◽  
Biolistic Delivery

AbstractAn important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.

scholarly journals Inactivation ofPhaeodactylum tricornutumurease gene using transcription activator-like effector nuclease-based targeted mutagenesis

2014 ◽  
Vol 13 (4) ◽  
pp. 460-470 ◽  
Author(s):  
Philip D. Weyman ◽  
Karen Beeri ◽  
Stephane C. Lefebvre ◽  
Josefa Rivera ◽  
James K. McCarthy ◽  
...  
Keyword(s):  
Targeted Mutagenesis ◽  
Transcription Activator

scholarly journals Targeted genome editing in Nicotiana tabacum using inducible CRISPR/Cas9 system

2020 ◽  
Author(s):  
Chong Ren ◽  
Yanfei Liu ◽  
Xida Wang ◽  
Yuchen Guo ◽  
Peige Fan ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Transgenic Plants ◽  
Genome Editing ◽  
Constitutive Expression ◽  
Phytoene Desaturase ◽  
Targeted Mutagenesis ◽  
Transcription Activator ◽  
Estradiol Treatment ◽  
Transgenic Lines ◽  
Pale Green

AbstractTargeted genome editing has been achieved in multiple plant species using the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system, in which the Cas9 gene is usually driven by constitutive promoters. However, constitutive expression of Cas9 is not necessary and can be harmful to plant development. In this study, we developed an estrogen-inducible CRISPR/Cas9 system by taking advantage of the chimeric transcription activator XVE and tested the efficacy of this inducible system in Nicotiana tabacum by targeting the phytoene desaturase (NtPDS) gene, whose mutation resulted in albino phenotypes. Treatment of four independent transgenic lines with exogenous estradiol successfully induced targeted mutagenesis in NtPDS. Sanger sequencing assay uncovered the presence of indel mutations (nucleotides insertions or deletions) at the target site as expected, and at least two types of mutations were identified for each line. Transgenic plants with mutated NtPDS gene after estradiol treatment exhibited pale green or incomplete albino leaves. Moreover, the expression of Cas9 in transgenic plants was strongly induced by estradiol treatment. Our results demonstrate the efficacy of XVE-based CRISPR/Cas9 system in N. tabacum, and the system reported here promises to be a useful approach for conditional genome editing, which would facilitate the study of genes of interest, especially those developmentally important genes.

Sign in / Sign up

Export Citation Format

Share Document