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.

scholarly journals Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley

2015 ◽  
Vol 5 (9) ◽  
pp. 1857-1863 ◽  
Author(s):  
Nagaveni Budhagatapalli ◽  
Twan Rutten ◽  
Maia Gurushidze ◽  
Jochen Kumlehn ◽  
Goetz Hensel
Keyword(s):  
Gene Function ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Homology Directed Repair

scholarly journals Poly(ADP-ribose) glycohydrolase promotes formation and homology-directed repair of meiotic DNA double-strand breaks independent of its catalytic activity

2020 ◽  
Author(s):  
Eva Janisiw ◽  
Marilina Raices ◽  
Fabiola Balmir ◽  
Luis Paulin Paz ◽  
Antoine Baudrimont ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Synaptonemal Complex ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Post Translational Modification ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Damage Repair ◽  
Somatic Tissues

SummaryPoly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in promoting the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.

scholarly journals Mutation in DDM1 inhibits the homology directed repair of double strand breaks

PLoS ONE ◽  
2019 ◽  
Vol 14 (2) ◽  
pp. e0211878 ◽  
Author(s):  
Seung Hee Choi ◽  
Tae Ho Ryu ◽  
Jeong-Il Kim ◽  
Sungbeom Lee ◽  
Seung Sik Lee ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Strand Breaks ◽  
Homology Directed Repair

scholarly journals Protein phosphatase PP6 is required for homology-directed repair of DNA double-strand breaks

Cell Cycle ◽  
2011 ◽  
Vol 10 (9) ◽  
pp. 1411-1419 ◽  
Author(s):  
Jianing Zhong ◽  
Ji Liao ◽  
Xin Liu ◽  
Pei Wang ◽  
Jinping Liu ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homology Directed Repair

scholarly journals Mitotic cells can repair DNA double-strand breaks via a homology-directed pathway

2020 ◽  
Vol 62 (1) ◽  
pp. 25-33
Author(s):  
Yuki Sakamoto ◽  
Tetsuya Kokuta ◽  
Ai Teshigahara ◽  
Kenta Iijima ◽  
Hiroyuki Kitao ◽  
...  
Keyword(s):  
Mitotic Cell ◽  
S Phase ◽  
Double Strand Breaks ◽  
G1 Phase ◽  
Dna Double Strand Breaks ◽  
Major Pathway ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Repair Efficiency ◽  
M Phase

Abstract The choice of repair pathways of DNA double-strand breaks (DSBs) is dependent upon the cell cycle phases. While homologous recombination repair (HRR) is active between the S and G2 phases, its involvement in mitotic DSB repair has not been examined in detail. In the present study, we developed a new reporter assay system to detect homology-directed repair (HDR), a major pathway used for HRR, in combination with an inducible DSB-generation system. As expected, the maximal HDR activity was observed in the late S phase, along with minimal activity in the G1 phase and at the G1/S boundary. Surprisingly, significant HDR activity was observed in M phase, and the repair efficiency was similar to that observed in late S phase. HDR was also confirmed in metaphase cells collected with continuous colcemid exposure. ChIP assays revealed the recruitment of RAD51 to the vicinity of DSBs in M phase. In addition, the ChIP assay for gamma-H2AX and phosphorylated DNA-PKcs indicated that a part of M-phase cells with DSBs could proceed into the next G1 phase. These results provide evidence showing that a portion of mitotic cell DSBs are undoubtedly repaired through action of the HDR repair pathway.

TALEN-mediated genome editing: prospects and perspectives

2014 ◽  
Vol 462 (1) ◽  
pp. 15-24 ◽  
Author(s):  
David A. Wright ◽  
Ting Li ◽  
Bing Yang ◽  
Martin H. Spalding
Keyword(s):  
Genome Editing ◽  
Double Strand Breaks ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Strand Breaks ◽  
Natural Processes ◽  
Current State ◽  
A Genome ◽  
Dna Dsbs ◽  
Repair Mechanisms

Genome editing is the practice of making predetermined and precise changes to a genome by controlling the location of DNA DSBs (double-strand breaks) and manipulating the cell's repair mechanisms. This technology results from harnessing natural processes that have taken decades and multiple lines of inquiry to understand. Through many false starts and iterative technology advances, the goal of genome editing is just now falling under the control of human hands as a routine and broadly applicable method. The present review attempts to define the technique and capture the discovery process while following its evolution from meganucleases and zinc finger nucleases to the current state of the art: TALEN (transcription-activator-like effector nuclease) technology. We also discuss factors that influence success, technical challenges and future prospects of this quickly evolving area of study and application.

scholarly journals The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks

Genes ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 1430
Author(s):  
Ian Yannuzzi ◽  
Margaret A. Butler ◽  
Joel Fernandez ◽  
Jeannine R. LaRocque
Keyword(s):  
Direct Repeat ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna End Resection ◽  
The Impact ◽  
End Resection

DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, we affirmed the importance of end resection on DSB repair pathway choice in multicellular systems, described the function of DmCtIP in short and extensive DNA end resection, and determined the impact of end resection on GCT length during HR.

scholarly journals Caffeine inhibits homology-directed repair of I-SceI-induced DNA double-strand breaks

Oncogene ◽  
2004 ◽  
Vol 23 (3) ◽  
pp. 824-834 ◽  
Author(s):  
Huichen Wang ◽  
Wilfried Boecker ◽  
Hongyan Wang ◽  
Xiang Wang ◽  
Jun Guan ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homology Directed Repair

scholarly journals Effects of mitoTALENs-Directed Double-Strand Breaks on Plant Mitochondrial Genomes

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 153
Author(s):  
Shin-ichi Arimura
Keyword(s):  
Double Strand Breaks ◽  
Mitochondrial Genomes ◽  
Transcription Activator ◽  
End Joining ◽  
Strand Breaks ◽  
Large Deletions ◽  
Naturally Occurring ◽  
Copy Numbers ◽  
Nuclear Genomes ◽  
Non Homologous End Joining

Mitochondrial genomes in flowering plants differ from those in animals and yeasts in several ways, including having large and variable sizes, circular, linear and branched structures, long repeat sequences that participate in homologous recombinations, and variable genes orders, even within a species. Understanding these differences has been hampered by a lack of genetic methods for transforming plant mitochondrial genomes. We recently succeeded in disrupting targeted genes in mitochondrial genomes by mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) in rice, rapeseed, and Arabidopsis. Double-strand breaks created by mitoTALENs were repaired not by non-homologous end-joining (NHEJ) but by homologous recombination (HR) between repeats near and far from the target sites, resulting in new genomic structures with large deletions and different configurations. On the other hand, in mammals, TALENs-induced DSBs cause small insertions or deletions in nuclear genomes and degradation of mitochondrial genomes. These results suggest that the mitochondrial and nuclear genomes of plants and mammals have distinct mechanisms for responding to naturally occurring DSBs. The different responses appear to be well suited to differences in size and copy numbers of each genome.

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