Repair of Site-Specific DNA Double-Strand Breaks in Barley Occurs via Diverse Pathways Primarily Involving the Sister Chromatid

Vu, Giang T. H.; Cao, Hieu X.; Watanabe, Koichi; Hensel, Goetz; Blattner, Frank R.; Kumlehn, Jochen; Schubert, Ingo

doi:10.1105/tpc.114.126607

Cited by 53 publications

(59 citation statements)

References 42 publications

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“…For OsAOX1c and Os BEL , short changes (≤ 3 bp) were the major type of mutation; conversely, all mutations in OsAOX1a were relatively long deletions. Because short deletions are often associated with classical NHEJ (cNHEJ) and longer deletions may represent the results of microhomology-mediated end-joining (or alternative NHEJ, aNHEJ)3738, the CRISPR/Cas9-induced mutation patterns might be different for specific DSB sites through distinguished NHEJ repair pathway.…”

Section: Resultsmentioning

confidence: 99%

Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system

Qin

et al. 2015

Sci Rep

200

125

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The CRISPR/Cas9 system is becoming an important genome editing tool for crop breeding. Although it has been demonstrated that target mutations can be transmitted to the next generation, their inheritance pattern has not yet been fully elucidated. Here, we describe the CRISPR/Cas9-mediated genome editing of four different rice genes with the help of online target-design tools. High-frequency mutagenesis and a large percentage of putative biallelic mutations were observed in T0 generations. Nonetheless, our results also indicate that the progeny genotypes of biallelic T0 lines are frequently difficult to predict and that the transmission of mutations largely does not conform to classical genetic laws, which suggests that the mutations in T0 transgenic rice are mainly somatic mutations. Next, we followed the inheritance pattern of T1 plants. Regardless of the presence of the CRISPR/Cas9 transgene, the mutations in T1 lines were stably transmitted to later generations, indicating a standard germline transmission pattern. Off-target effects were also evaluated, and our results indicate that with careful target selection, off-target mutations are rare in CRISPR/Cas9-mediated rice gene editing. Taken together, our results indicate the promising production of inheritable and “transgene clean” targeted genome-modified rice in the T1 generation using the CRISPR/Cas9 system.

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Section: Resultsmentioning

confidence: 99%

Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system

Qin

et al. 2015

Sci Rep

200

125

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“…In addition, many inserted sequences at I-SceI break sites have been shown to be templated from sequences near the DSB sites and to be rejoined via MMEJ; therefore, a model termed synthesisdependent MMEJ has been proposed (Yu and McVey, 2010). On the other hand, it has been demonstrated that sequences from elsewhere in the genome or around the DSB site may be inserted into I-SceI break sites in exogenous genes via a synthesisdependent strand annealing (SDSA)-like model in tobacco (Salomon and Puchta, 1998) and barley (Vu et al, 2014). These findings have suggested that the SDSA-like model may result in insertions within DSB sites by homology search via microhomology and copying from sister chromatid DNA.…”

Section: Discussionmentioning

confidence: 99%

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice

et al. 2015

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We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.

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“…These differences suggest that the barley genome is still quite dynamic. In particular, the frequent occurrence of INDELS in non-coding regions point to the importance of DNA damage repair by erroneous pathways in barley, as suggested (Vu et al, 2014). In addition, the high degree of sequence divergence between cultivars could have impacted gene targeting efficiencies if isogenic ALS gene DNA had not been used.…”

Section: Discussionmentioning

confidence: 83%

Gene Targeting Without DSB Induction Is Inefficient in Barley

2017

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Double strand-break (DSB) induction allowed efficient gene targeting in barley (Hordeum vulgare), but little is known about efficiencies in its absence. To obtain such data, an assay system based on the acetolactate synthase (ALS) gene was established, a target gene which had been used previously in rice and Arabidopsis thaliana. Expression of recombinases RAD51 and RAD54 had been shown to improve gene targeting in A. thaliana and positive-negative (P-N) selection allows the routine production of targeted mutants without DSB induction in rice. We implemented these approaches in barley and analysed gene targeting with the ALS gene in wild type and RAD51 and RAD54 transgenic lines. In addition, P-N selection was tested. In contrast to the high gene targeting efficiencies obtained in the absence of DSB induction in A. thaliana or rice, not one single gene targeting event was obtained in barley. These data suggest that gene targeting efficiencies are very low in barley and can substantially differ between different plants, even at the same target locus. They also suggest that the amount of labour and time would become unreasonably high to use these methods as a tool in routine applications. This is particularly true since DSB induction offers efficient alternatives. Barley, unlike rice and A. thaliana has a large, complex genome, suggesting that genome size or complexity could be the reason for the low efficiencies. We discuss to what extent transformation methods, genome size or genome complexity could contribute to the striking differences in the gene targeting efficiencies between barley, rice and A. thaliana.

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Repair of Site-Specific DNA Double-Strand Breaks in Barley Occurs via Diverse Pathways Primarily Involving the Sister Chromatid

Cited by 53 publications

References 42 publications

Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system

Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice

Gene Targeting Without DSB Induction Is Inefficient in Barley

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