Targeted molecular trait stacking in cotton through targeted double‐strand break induction

D’Halluin, Kathleen; Vanderstraeten, Chantal; Hulle, Jolien Van; Rosolowska, Joanna; Brande, Ilse van den; Pennewaert, Anouk; D'Hont, Kristel; Bossut, Martine; Jantz, Derek; Ruiter, R.K.; Broadhvest, Jean

doi:10.1111/pbi.12085

Cited by 137 publications

(89 citation statements)

References 45 publications

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“…HDR brings other opportunities in genome editing but has been more challenging. As a result, reports describing successful gene editing or site-specific trait gene integration through HDR in plants are limited (Cai et al, 2009;Shukla et al, 2009;Townsend et al, 2009;Ainley et al, 2013;D'Halluin et al, 2013;Li et al, 2013;Shan et al, 2013;Zhang et al, 2013).…”

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confidence: 99%

Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

et al. 2015

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Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.

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confidence: 99%

Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

et al. 2015

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“…This repair pathway is not as efficient as the non-homologous end-joining pathway, but success has been reported for ZFN and meganuclease-mediated integration of a new DNA fragment into a preexisting transgenic locus in major crop plants. 11,12 This demonstrates their potential use for repeated gene integration at the same genomic location.…”

Section: Introductionmentioning

confidence: 86%

Precise, flexible and affordable gene stacking for crop improvement

Chen

2017

Bioengineered

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The genetic engineering of plants offers a revolutionary advance for crop improvement, and the incorporation of transgenes into crop species can impart new traits that would otherwise be difficult to obtain through conventional breeding. Transgenes introduced into plants, however, can only be useful when bred out to field cultivars. As new traits are continually added to further improve transgenic cultivars, clustering new DNA near previously introduced transgenes keep from inflating the number of segregating units that breeders must assemble back into a breeding line. Here we discuss various options to introduce DNA site-specifically into an existing transgenic locus. As food security is becoming a pressing global issue, the old proverb resonates true to this day: "give a man a fish and you feed him for a day; teach a man to fish and you feed him for a lifetime." Hence, we describe a recombinase-mediate gene stacking system designed with freedom to operate, providing an affordable option for crop improvement by less developed countries where food security is most at risk.

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“…The tangibility of targeted gene stacking in cotton by means of adopting specifically engineered mega nucleases has been reported (D'Halluin et al, 2013). In these experiments, gene present in the embryogenic cells of cotton possessing a site adjoining to an insect persistence has been targeted to promote double stranded breaks via homologous recombination in the presence of DNA template possessing two different genes for herbicide resistance flanked by DNA arrays with homology to the target site.…”

Section: Cottonmentioning

confidence: 99%