scholarly journals Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting

2008 ◽  
Vol 36 (19) ◽  
pp. 6333-6342 ◽  
Author(s):  
Susumu Iiizumi ◽  
Aya Kurosawa ◽  
Sairei So ◽  
Yasuyuki Ishii ◽  
Yuichi Chikaraishi ◽  
...  
Keyword(s):  
Gene Targeting ◽  
End Joining ◽  
Dna Integration ◽  
Non Homologous End Joining

scholarly journals Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Qi Jia ◽  
Paul Bundock ◽  
Paul J. J. Hooykaas ◽  
Sylvia de Pater
Keyword(s):  
Gene Targeting ◽  
Wild Type ◽  
End Joining ◽  
Dsb Repair ◽  
Critical Pathway ◽  
Dna Integration ◽  
Dna Insertion ◽  
Non Homologous End Joining ◽  
The Stability

In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

scholarly journals Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

2021 ◽  
Author(s):  
Daisuke Miki ◽  
Rui Wang ◽  
Jing Li ◽  
Dali Kong ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Agronomic Traits ◽  
Higher Plants ◽  
Crop Improvement ◽  
Strand Break ◽  
End Joining ◽  
Homology Directed Repair ◽  
Target Dna ◽  
Targeted Gene Editing ◽  
Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

Efficient gene targeting in non-homologous end-joining-deficient Lipomyces starkeyi strains

2017 ◽  
Vol 63 (4) ◽  
pp. 751-763 ◽  
Author(s):  
Yoshifumi Oguro ◽  
Harutake Yamazaki ◽  
Satoshi Ara ◽  
Yosuke Shida ◽  
Wataru Ogasawara ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Lipomyces Starkeyi ◽  
End Joining ◽  
Efficient Gene ◽  
Non Homologous End Joining

Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining

2008 ◽  
Vol 45 (1) ◽  
pp. 68-75 ◽  
Author(s):  
François Villalba ◽  
Jérôme Collemare ◽  
Patricia Landraud ◽  
Karine Lambou ◽  
Viviane Brozek ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Magnaporthe Grisea ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals Homology-directed repair of a defective glabrous gene in Arabidopsis with Cas9-based gene targeting

2018 ◽  
Author(s):  
Florian Hahn ◽  
Marion Eisenhut ◽  
Otho Mantegazza ◽  
Andreas P.M. Weber
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Strand Break ◽  
Double Strand Break ◽  
In Planta ◽  
End Joining ◽  
Trichome Formation ◽  
Break Site ◽  
Repair Template ◽  
Non Homologous End Joining

ABSTRACTThe CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell’s own repair machinery either by the non-homologous end joining pathway or by homologous recombination. While the first repair mechanism results in random mutations at the double-strand break site, homologous recombination uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by homologous recombination are very low. In this study, we compared two methods that have been reported to enhance frequencies of homologous recombination in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of homologous recombination events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by homologous recombination at frequencies of 0.12% using the in planta gene targeting approach, while both approaches using viral replicons did not yield any trichome-bearing plants.

scholarly journals CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Li Zhu ◽  
Hiroaki Mon ◽  
Jian Xu ◽  
Jae Man Lee ◽  
Takahiro Kusakabe
Keyword(s):  
Gene Targeting ◽  
End Joining ◽  
Non Homologous End Joining

T-DNA integration in plants requires MRE11- or TDP2-mediated removal of the 5’ bound Agrobacterium protein VirD2

2021 ◽  
Author(s):  
Lejon Kralemann ◽  
Sylvia de Pater ◽  
Hexi Shen ◽  
Susan Kloet ◽  
Robin van Schendel ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Mechanistic Model ◽  
Somatic Tissue ◽  
Plant Genome ◽  
Bacterial Protein ◽  
End Joining ◽  
Plant Genetic Engineering ◽  
Dna Integration ◽  
Dna Molecule ◽  
Non Homologous End Joining

Abstract Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides T-DNA into the plants nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA’s 5’ end. Different from capture of the 3’ end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5’ attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5’-end blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5’ end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3’ capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant’s own DNA repair machineries to transform them.

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