Double-Strand Break Repair and Its Application to Genome Engineering in Plants

Puchta, Holger; Fauser, Friedrich

doi:10.1007/978-1-4939-2556-8_1

Cited by 9 publications

(9 citation statements)

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“…DSBR is thought to be the major DSBR pathway during meiosis, and its limitation during somatic recombination prevents crossover frequencies from becoming too high, which would result in di‐ and acentric chromosomes. This is due to multiple ectopic homologies in the plant genome being used as templates for HR, which can be (i) intrachromosomal homologies, (ii) the sister chromatid, (iii) allelic sequences in diploid cells, or (iv) homologous sequences in ectopic positions (Puchta and Fauser, ). The SDSA model supports the explanation of observed gene targeting (GT) experiments in somatic plant cells.…”

Section: Dna Repair Pathways Utilized For Genome Editingmentioning

confidence: 99%

From classical mutagenesis to nuclease‐based breeding – directing natural DNA repair for a natural end‐product

2017

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Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases (SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based 'DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants.

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Section: Dna Repair Pathways Utilized For Genome Editingmentioning

confidence: 99%

From classical mutagenesis to nuclease‐based breeding – directing natural DNA repair for a natural end‐product

2017

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“…Protoplasts can be transformed with both SSNs and DNA repair template at high efficiency (Wright et al, 2005; Townsend et al, 2009; Zhang et al, 2013), however, for most plant species, especially major cereal crops, regeneration of plants from cultured protoplasts is still not feasible. An efficient way to supply the pant cell with a matrix for HDR-mediated DSB repair is to use an incoming T-DNA from Agrobacterium tumefaciens or transfected plasmid DNA (Puchta and Fauser, 2015). A sophisticated system, in planta GT, to enhance gene replacement was first established in 2012 using the meganuclease I-SceI (Fauser et al, 2012), then successfully applied in Arabidopsis using the CRISPR/Cas9 reagent by the same group in 2014 (Schiml et al, 2014).…”

Section: Challenges and Future Perspectivesmentioning

confidence: 99%

“…In planta GT system allows the simultaneous release of a linear GT vector and the induction of a DSB at the target locus. Under this strategy, the GT vector can be designed for the site-specific integration of transgenes or to modify the target gene/locus in a predefined manner (Puchta and Fauser, 2015). Considering the low DNA titers delivered by Agrobacterium -mediated gene transfer, biolistic gene transfer may be superior for HDR-mediated GT by simultaneously delivery of both SSNs and DNA repair template and providing larger quantities of repair template.…”

Section: Challenges and Future Perspectivesmentioning

confidence: 99%

“…For crop species recalcitrant to transformation and regeneration, intra-chromosome HDR will be an effective alternative strategy (Kumar et al, 2016). Furthermore, suppression of core components of the NHEJ pathway or enhancement of key elements of HDR machinery can be used to increase frequencies of GT (Puchta et al, 1996; Qi et al, 2013; Puchta and Fauser, 2015). In addition, to date, most of the GT events reported in plants were either herbicide resistant genes or selectable marker genes in which positive selection for GT is much easier such that precise insertion or gene replacement of the donor in the target locus conferred resistance to selection and avoidance of random integration of the donor ( Table 1 ).…”

Section: Challenges and Future Perspectivesmentioning

confidence: 99%

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Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement

Sun

Xia

2016

Front. Plant Sci.

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Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired “safe” harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in plant cells. We then address the challenges and propose future perspectives in order to facilitate the implementation of precise genome modification through SSNs-mediated GT for crop improvement in a global context.

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“…Each Cas9-induced DSB produces two free ends that become actively managed by the plant's own elaborate DSB repair processes. The plant typically reconnects the two cut ends through the Nonhomologous End Joining (NHEJ) DSB repair pathway 1,22,23 . The cut and repair process may go through many cycles until the religated ends are mutated, preventing recognition and cutting by the Cas9-sgRNA complex 14 .…”

Section: Introductionmentioning

confidence: 99%

Translocation and duplication from CRISPR-Cas9 editing inArabidopsis thaliana

Inagaki

et al. 2018

Preprint

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Cut DNA ends in plants may recombine to form novel molecules. We asked whether CRISPR-Cas9 expression in plants could induce nonhomologous recombination between diverse and heterologous broken DNA ends. We induced two breaks separated by 2.3 or by 8.5 kilobases leading to duplication of the intervening DNA and meiotic transmission of the 2.3kb duplication. Two or more dsDNA breaks in nonhomologous chromosomes led to ligation of breakpoints consistent with chromosome arm translocations. Screening 881 primary transformants we obtained 195 PCR products spanning independent, expected translocation junctions involving ends produced by cutting different loci. Sequencing indicated a true positive rate of 84/91 and demonstrated the occurrence of different junction alleles. A majority of the resulting structures would be deleterious and none were transmitted meiotically. Ligation of interchromosomal, heterologous dsDNA ends suggest that the CRISPR-Cas9 can be used to engineer plant genes and chromosomes in vivo.Significance StatementWe explored how genome editing tools such as CRISPR-Cas9 could provide new ways to tailor novel genomic combinations and arrangements. We show that distant cut ends often precisely come together, that cuts in different chromosomes can result in translocations, and that two cuts within a chromosome often result in the duplication of the intervening segment. Formation of multiple structures with precise junctions will enable engineered rearrangements that can be predicted with accuracy.

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Double-Strand Break Repair and Its Application to Genome Engineering in Plants

Cited by 9 publications

References 78 publications

From classical mutagenesis to nuclease‐based breeding – directing natural DNA repair for a natural end‐product

From classical mutagenesis to nuclease‐based breeding – directing natural DNA repair for a natural end‐product

Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement

Translocation and duplication from CRISPR-Cas9 editing inArabidopsis thaliana

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