Approaches for gene targeting and targeted gene expression in plants

Husaini, Amjad M.; Rashid, Zerka; Mir, Reyazul Rouf; Aquil, Bushra

doi:10.4161/gmcr.2.3.18605

Cited by 16 publications

(6 citation statements)

References 205 publications

(183 reference statements)

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“…Through directed co-localization and dimerization of two FokI nuclease monomers, ZFNs generate a functional site-specific endonuclease that creates a DSB at the targeted locus (Mani et al, 2005). Through the use of this methodology, the induced DNA sequence modifications can range from mutations to gene replacement, site-specific structural changes, or gene insertion, to name a few (Husaini et al, 2011). …”

Section: Introductionmentioning

confidence: 99%

Identification of â€œsafe harborâ€ loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair

et al. 2014

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Zinc-finger nucleases (ZFNs) have proved to be successful tools for targeted genome manipulation in several organisms. Their main property is the induction of double-strand breaks (DSBs) at specific sites, which are further repaired through homologous recombination (HR) or non-homologous end joining (NHEJ). However, for the appropriate integration of genes at specific chromosomal locations, proper sites for gene integration need to be identified. These regions, hereby named safe harbor loci, must be localized in non-coding regions and possess high gene expression. In the present study, three different ZFN constructs (pZFN1, pZFN2, pZFN3), harboring β-glucuronidase (GUS) as a reporter gene, were used to identify safe harbor loci on rice chromosomes. The constructs were delivered into IR64 rice by using an improved Agrobacterium-mediated transformation protocol, based on the use of immature embryos. Gene expression was measured by histochemical GUS activity and the flanking regions were determined through thermal-asymmetric interlaced polymerase chain reaction (TAIL PCR). Following sequencing, 28 regions were identified as putative sites for safe integration, but only one was localized in a non-coding region and also possessed high GUS expression. These findings have significant applicability to create crops with new and valuable traits, since the site can be subsequently used to stably introduce one or more genes in a targeted manner.

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Section: Introductionmentioning

confidence: 99%

Identification of â€œsafe harborâ€ loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair

et al. 2014

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“…e . site of the transgene insertion on the chromosome) 86 , 87 resulting in reduced transcription of the protein as compared to the ER-targeting lines. Generally, recombinant proteins targeted to the cytosol are detected at very low levels despite high mRNA levels, resulting in accumulation rates below 0.1% of total soluble protein (TSP) in several cases 88 , 89 .…”

Section: Discussionmentioning

confidence: 99%

Reducing biomass recalcitrance by heterologous expression of a bacterial peroxidase in tobacco (Nicotiana benthamiana)

Ligaba-Osena

Hankoua

DiMarco

et al. 2017

Sci Rep

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Commercial scale production of biofuels from lignocellulosic feed stocks has been hampered by the resistance of plant cell walls to enzymatic conversion, primarily owing to lignin. This study investigated whether DypB, the lignin-degrading peroxidase from Rodococcus jostii, depolymerizes lignin and reduces recalcitrance in transgenic tobacco (Nicotiana benthamiana). The protein was targeted to the cytosol or the ER using ER-targeting and retention signal peptides. For each construct, five independent transgenic lines were characterized phenotypically and genotypically. Our findings reveal that expression of DypB in the cytosol and ER does not affect plant development. ER-targeting increased protein accumulation, and extracts from transgenic leaves showed higher activity on classic peroxidase substrates than the control. Intriguingly, in situ DypB activation and subsequent saccharification released nearly 200% more fermentable sugars from transgenic lines than controls, which were not explained by variation in initial structural and non-structural carbohydrates and lignin content. Pyrolysis-GC-MS analysis showed more reduction in the level of lignin associated pyrolysates in the transgenic lines than the control primarily when the enzyme is activated prior to pyrolysis, consistent with increased lignin degradation and improved saccharification. The findings reveal for the first time that accumulation and in situ activation of a peroxidase improves biomass digestibility.

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“…In the first case, the aim is to create localised, singlepoint mutations, whereas the existing target sequence is substituted with another one in the second case (Carroll 2011). While gene targeting is routinely applied in yeast and mice (Rothstein 1983;Capecchi 2005), and is rather advanced in moss Physcomitrella (Roberts et al 2011), its application in crops has been difficult (Husaini et al 2011). Two recently introduced approaches, the zinc-finger nuclease (ZFN) (Carroll 2011) and the transcription activator-like (TAL) effector proteins (Bogdanove and Voytas 2011), exploit enzymatic complexes acting on DNA.…”

Section: Gene Targetingmentioning

confidence: 99%

Induced Genetic Variation, TILLING and NGS-Based Cloning

Salvi

Druka

Milner

et al. 2014

Biotechnological Approaches to Barley Improvement

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Approaches for gene targeting and targeted gene expression in plants

Cited by 16 publications

References 205 publications

Identification of â€œsafe harborâ€ loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair

Identification of â€œsafe harborâ€ loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair

Reducing biomass recalcitrance by heterologous expression of a bacterial peroxidase in tobacco (Nicotiana benthamiana)

Induced Genetic Variation, TILLING and NGS-Based Cloning

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Approaches for gene targeting and targeted gene expression in plants

Cited by 16 publications

References 205 publications

Identification of â€œsafe harborâ€ loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair

Identification of â€œsafe harborâ€ loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair

Reducing biomass recalcitrance by heterologous expression of a bacterial peroxidase in tobacco (Nicotiana benthamiana)

Induced Genetic Variation, TILLING and NGS-Based Cloning

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Product

Resources

About

Identification of â€œsafe harborâ€ loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair

Identification of â€œsafe harborâ€ loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair