DNA methylation occurring in Cre‐expressing cells inhibits loxP recombination and silences loxP‐sandwiched genes

Liu, Ruochen; Qin, Long; Zou, Xin; Wang, You; Pei, Yan

doi:10.1111/nph.17353

Cited by 8 publications

(5 citation statements)

References 98 publications

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“…Serine integrases, in contrast, use two distinct sites, leading to a directional and irreversible recombination event. Another limitation on the use of tyrosine integrases in plants has been low efficiency and silencing, which has recently been connected to CHH methylation of tyrosine recombinase sites 31 .…”

Section: Introductionmentioning

confidence: 99%

An integrase toolbox to record gene-expression during plant development

et al. 2023

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There are many open questions about the mechanisms that coordinate the dynamic, multicellular behaviors required for organogenesis. Synthetic circuits that can record in vivo signaling networks have been critical in elucidating animal development. Here, we report on the transfer of this technology to plants using orthogonal serine integrases to mediate site-specific and irreversible DNA recombination visualized by switching between fluorescent reporters. When combined with promoters expressed during lateral root initiation, integrases amplify reporter signal and permanently mark all descendants. In addition, we present a suite of methods to tune the threshold for integrase switching, including: RNA/protein degradation tags, a nuclear localization signal, and a split-intein system. These tools improve the robustness of integrase-mediated switching with different promoters and the stability of switching behavior over multiple generations. Although each promoter requires tuning for optimal performance, this integrase toolbox can be used to build history-dependent circuits to decode the order of expression during organogenesis in many contexts.

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Section: Introductionmentioning

confidence: 99%

An integrase toolbox to record gene-expression during plant development

et al. 2023

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“…However, the same plants were not activated under copper treatment. A possible explanation for this behavior is the posttranslational gene silencing of the flippase transgene, as earlier reported for tyrosine integrases such as Flp operating in transgenic plants (Guiziou et al, 2023; R. Liu et al, 2021).…”

Section: Discussionmentioning

confidence: 64%

CuBe: a geminivirus-based copper-regulated expression system suitable for post-harvest activation

Garcia-Perez,

Vazquez-Vilar,

Lozano-Duran

et al. 2024

Preprint

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The growing demand for sustainable platforms for biomolecule manufacturing has fueled the development of plant-based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large-scale production due to high operational costs and batch-to-batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)-derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulfate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harboring the gene of interest (GOI) flanked by cis-acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper-inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast-derived CUP2-Gal4 copper-responsive transcriptional activator for Rep/RepA expression, and (iv) a copper-inducible Flp recombinase to minimize basal Rep/RepA expression. Copper sulfate application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti-SARS-CoV-2 antibody upon copper treatment. Notably, the system is also functional with post-harvest copper application, a strategy with potential advantages for large-scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost-effective and scalable production of recombinant proteins in plants.

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“…In addition, the massive immune response, extreme genetic diversity, and strong epigenetic/ontogenetic variation discussed above warrant investigation into their biological mechanisms and variability. Finally, some of the basic technologies required for advanced rDNA‐based modification, such as that of gene excision, appear to work very poorly or not at all in trees, likely a reflection of the extensive DNA methylation that accompanies recombinase induction (Liu et al ., 2021). The desire to remove any transformation‐promoting genes from the genome to avoid pleiotropy, or to remove gene‐editing reagents to ease regulatory acceptance and reduce off‐target mutagenesis, will require smarter excision systems as well as much improved transformation technologies.…”

Section: Science and Technology Innovationmentioning

confidence: 99%

Social and biological innovations are essential to deliver transformative forest biotechnologies

Boerjan,

Strauss

2024

New Phytologist

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SummaryForests make immense contributions to societies in the form of ecological services and sustainable industrial products. However, they face major challenges to their viability and economic use due to climate change and growing biotic and economic threats, for which recombinant DNA (rDNA) technology can sometimes provide solutions. But the application of rDNA technologies to forest trees faces major social and biological obstacles that make its societal acceptance a ‘wicked’ problem without straightforward solutions. We discuss the nature of these problems, and the social and biological innovations that we consider essential for progress. As case studies of biological challenges, we focus on studies of modifications in wood chemistry and transformation efficiency. We call for major innovations in regulations, and the dissolution of method‐based market barriers, that together could lead to greater research investments, enable wide use of field studies, and facilitate the integration of rDNA‐modified trees into conventional breeding programs. Without near‐term adoption of such innovations, rDNA‐based solutions will be largely unavailable to help forests adapt to the growing stresses from climate change and the proliferation of forest pests, nor will they be available to provide economic and environmental benefits from expanded use of wood and related bioproducts as part of an expanding bioeconomy.

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DNA methylation occurring in Cre‐expressing cells inhibits loxP recombination and silences loxP‐sandwiched genes

Cited by 8 publications

References 98 publications

An integrase toolbox to record gene-expression during plant development

An integrase toolbox to record gene-expression during plant development

CuBe: a geminivirus-based copper-regulated expression system suitable for post-harvest activation

Social and biological innovations are essential to deliver transformative forest biotechnologies

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