A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA

Nishizawa‐Yokoi, Ayako; Nonaka, Satoko; Osakabe, Keishi; Saika, Hiroaki; Toki, Seiichi

doi:10.1104/pp.15.00638

Cited by 20 publications

(15 citation statements)

References 42 publications

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“…The positive/negative system using the DT-A subunit might have posed risks to dicots, because it has not been successfully applied in those plants. Therefore, an alternative positive/negative selection system was developed as an alternative, based on a caffeic acid O-methyltransferase (codA) D314A single-mutated version as the negative selection marker (Osakabe et al 2014) or neomycin phosphotransferase II (NptII) (positive)/RNAi-based anti-NptII (negative) selection at much lower frequencies (Nishizawa-Yokoi et al 2015b), which might be a result of less efficient G418 selection in rice. Nonetheless, the positive/negative selection strategy was shown to be unsuccessful in barley (Horvath et al 2016), highlighting its extremely low efficiency in the absence of DSB and the high genome complexity of monocot gene targeting.…”

Section: Reviewmentioning

confidence: 99%

Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants

Sung

Kim

et al. 2019

Rice

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Continuing crop domestication/redomestication and modification is a key determinant of the adaptation and fulfillment of the food requirements of an exploding global population under increasingly challenging conditions such as climate change and the reduction in arable lands. Monocotyledonous crops are not only responsible for approximately 70% of total global crop production, indicating their important roles in human life, but also the first crops to be challenged with the abovementioned hurdles; hence, monocot crops should be the first to be engineered and/or de novo domesticated/redomesticated. A long time has passed since the first green revolution; the world is again facing the challenge of feeding a predicted 9.7 billion people in 2050, since the decline in world hunger was reversed in 2015. One of the major lessons learned from the first green revolution is the importance of novel and advanced trait-carrying crop varieties that are ideally adapted to new agricultural practices. New plant breeding techniques (NPBTs), such as genome editing, could help us succeed in this mission to create novel and advanced crops. Considering the importance of NPBTs in crop genetic improvement, we attempt to summarize and discuss the latest progress with major approaches, such as site-directed mutagenesis using molecular scissors, base editors and especially homology-directed gene targeting (HGT), a very challenging but potentially highly precise genome modification approach in plants. We therefore suggest potential approaches for the improvement of practical HGT, focusing on monocots, and discuss a potential approach for the regulation of genome-edited products.

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Section: Reviewmentioning

confidence: 99%

Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants

Sung

Kim

et al. 2019

Rice

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“…Luciferase luminescence was detected in these transgenic lines (Figure S2). In our previous studies, it was impossible to eliminate completely transgenic calli in which T-DNA had integrated randomly into the host genome even with positive-negative selection with an extraordinarily toxic peptide, DT-A, as a negative selection marker (Nishizawa-Yokoi et al, 2015a, 2015b. Using positive-negative selection, 99% of hygromycin-resistant calli was thought to be transgenic calli with random T-DNA integration because of partial T-DNA integration (i.e.…”

Section: Piggybac-mediated Temporary Transgenesis Of Reporter Gene In Ricementioning

confidence: 99%

A piggyBac‐mediated transgenesis system for the temporary expression of CRISPR/Cas9 in rice

Nishizawa‐Yokoi

Toki

2021

Plant Biotechnology Journal

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Targeted mutagenesis via CRISPR/Cas9 is now widely used, not only in model plants but also in agriculturally important crops. However, in vegetative crop propagation, CRISPR/Cas9 expression cassettes cannot be segregated out in the resulting progenies, but must nevertheless be eliminated without leaving unnecessary sequences in the genome. To this end, we designed a piggyBac-mediated transgenesis system for the temporary expression of CRISPR/Cas9 in plants. This system allows integration into the host genome of piggyBac carrying both CRISPR/Cas9 and positive selection marker expression cassettes from an extrachromosomal double-stranded transfer DNA (dsT-DNA), with subsequent excision of the transgenes by the re-transposition of piggyBac from the host genome after successful induction of targeted mutagenesis via CRISPR/ Cas9. Here, we demonstrate that the transgenesis system via piggyBac transposition from T-DNA works to deliver transgenes in rice. Following positive-negative selection to exclude transgenic cells randomly transformed with T-DNA, piggyBac-mediated transgenesis from the extrachromosomal dsT-DNA was successful in ca. 1% of transgenic callus lines. After temporary expression of CRISPR/Cas9 within piggyBac, we confirmed, in a proof-of-concept experiment, that piggyBac could be excised precisely from the genome via the stably transformed transposase PBase. Even after excision of piggyBac, CRISPR/Cas9-induced targeted mutations could be detected in the endogenous gene in regenerated rice plants. These results suggest that our piggyBac-mediated transgenesis system will be a valuable tool in establishing efficient CRISPR/ Cas9-mediated targeted mutagenesis in vegetatively propagated crops.

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“…Ever since HDR was demonstrated to be feasible in plant cells in 1988 (Paszkowski et al, 1988), various classical GT strategies have been attempted to achieve HDR in plants (Paszkowski et al, 1988; Offringa et al, 1990; Zhu et al, 2000; Terada et al, 2002; Okuzaki and Toriyama, 2004; Shaked et al, 2005; Nishizawa-Yokoi et al, 2015). The induction of a DSB at a specific locus can significantly increase the frequency of homologous recombination up to more than 100-fold (Puchta et al, 1996).…”

Section: Ssns-mediated Gene Targeting In Plantsmentioning

confidence: 99%

“…Since then, the endogenous rice genes at more than 10 loci have been targeted (Terada et al, 2007; Yamauchi et al, 2009; Moritoh et al, 2012; Ono et al, 2012; Ozawa et al, 2012; Dang et al, 2013; Osakabe et al, 2014). Using a combination of neomycin phosphotransferase II ( nptII ) and an antisense nptII construct, a universally applicable PNS system for GT in plants was established, although negative selection with this system is relatively less efficient compared with DT-A (Nishizawa-Yokoi et al, 2015). It is worth to note that although this strategy has been only documented in classical GT experiment, it is expected that combination of SSNs-mediated GT with PNS strategy may facilitate the enrichment and recovery of GT events in plants.…”

Section: Challenges and Future Perspectivesmentioning

confidence: 99%

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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A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA

Cited by 20 publications

References 42 publications

Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants

Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants

A piggyBac‐mediated transgenesis system for the temporary expression of CRISPR/Cas9 in rice

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

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