Use of the Cas9 Orthologs from Streptococcus thermophilus and Staphylococcus aureus for Non-Homologous End-Joining Mediated Site-Specific Mutagenesis in Arabidopsis thaliana

2017 ◽  
pp. 365-376 ◽  
Author(s):  
Jeannette Steinert ◽  
Carla Schmidt ◽  
Holger Puchta
Keyword(s):  
Staphylococcus Aureus ◽  
Arabidopsis Thaliana ◽  
Streptococcus Thermophilus ◽  
End Joining ◽  
Site Specific ◽  
Non Homologous End Joining ◽  
And Staphylococcus Aureus

Editing livestock genomes with site-specific nucleases

2014 ◽  
Vol 26 (1) ◽  
pp. 74 ◽  
Author(s):  
Daniel F. Carlson ◽  
Wenfang Tan ◽  
Perry B. Hackett ◽  
Scott C. Fahrenkrug
Keyword(s):  
Genetic Alterations ◽  
Large Animal ◽  
Dna Breaks ◽  
Transcription Activator ◽  
End Joining ◽  
Site Specific ◽  
Homology Directed Repair ◽  
Double Strand Dna Breaks ◽  
Non Homologous End Joining ◽  
Inactive Genes

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

Poly(ADP-ribose)polymerases are involved in microhomology mediated back-up non-homologous end joining in Arabidopsis thaliana

2013 ◽  
Vol 82 (4-5) ◽  
pp. 339-351 ◽  
Author(s):  
Qi Jia ◽  
Amke den Dulk-Ras ◽  
Hexi Shen ◽  
Paul J. J. Hooykaas ◽  
Sylvia de Pater
Keyword(s):  
Arabidopsis Thaliana ◽  
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.

scholarly journals Inhibition of NHEJ repair by type II-A CRISPR-Cas systems

2017 ◽  
Author(s):  
Aude Bernheim ◽  
Alicia Calvo Villamanan ◽  
Clovis Basier ◽  
Eduardo PC Rocha ◽  
Marie Touchon ◽  
...  
Keyword(s):  
Genetic Material ◽  
Streptococcus Thermophilus ◽  
Double Strand Breaks ◽  
Type Ii ◽  
End Joining ◽  
Bacterial Genomes ◽  
Strand Breaks ◽  
Nhej Repair ◽  
Repair Mechanisms ◽  
Non Homologous End Joining

AbstractCRISPR-Cas systems introduce double strand breaks into DNA of invading genetic material and use DNA fragments to acquire novel spacers during adaptation. Double strand breaks are the substrate of several bacterial DNA repair pathways, paving the way for interactions between them and CRISPR-Cas systems. Here, we hypothesized that non-homologous end joining (NHEJ) interferes with type II CRISPR-Cas systems. We tested this idea by studying the patterns of co-occurrence of the two systems in bacterial genomes. We found that NHEJ and type II-A CRISPR-Cas systems only co-occur once among 5563 fully sequenced prokaryotic genomes. We investigated experimentally the possible molecular interactions causing this negative association using the NHEJ pathway from Bacillus subtilis and the type II-A CRISPR-Cas systems from Streptococcus thermophilus and Streptococcus pyogenes. Our results suggest that the NHEJ system has no effect on type II-A CRISPR-Cas interference and adaptation. On the other hand, we provide evidence for the inhibition of NHEJ repair by the Csn2 protein from type II-A CRISPR-Cas system. Our findings give insights on the complex interactions between CRISPR- Cas systems and repair mechanisms in bacteria and contribute to explain the scattered distribution of CRISPR-Cas systems in bacterial genomes.

Sign in / Sign up

Export Citation Format

Share Document