External Publications

Plant Artificial Chromosome Technology

AMELIA FRIZELL-ARMITAGE of The Global Plant Council introduces future directions for Plant Artificial Chromosome Technology:

Plant artificial chromosomes (PACs) have many advantages over traditional transformation systems. For example, to confer complex traits such as disease resistance and tolerance to abiotic stresses such as heat and drought, multiple genes are required. This is not easy with current methods of modification.

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There's also mention of the recent workshop on the CRISPR-CAS system of genome editing held in September 2015 by GARNet and OpenPlant at the John Innes Centre in Norwich! You can read the full meeting report here.

A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard

A new publication from Diego Orzaez's lab demonstrates the functionality and the efficiency of gRNA–Cas9 GB tools in Nicotiana benthamiana - a useful toolbox for plant synthetic biology! Vazquez-Vilar, M., Bernabé-Orts, J.M., Fernandez-del-Carmen, A., Ziarsolo, P., Blanca, J., Granell, A. and Orzaez, D., 2016. A modular toolbox for gRNA–Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods, 12(1), p.1.

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Abstract

Background

The efficiency, versatility and multiplexing capacity of RNA-guided genome engineering using the CRISPR/Cas9 technology enables a variety of applications in plants, ranging from gene editing to the construction of transcriptional gene circuits, many of which depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell. The engineering principles of standardization and modularity applied to DNA cloning are impacting plant genetic engineering, by increasing multigene assembly efficiency and by fostering the exchange of well-defined physical DNA parts with precise functional information.

Results

Here we describe the adaptation of the RNA-guided Cas9 system to GoldenBraid (GB), a modular DNA construction framework being increasingly used in Plant Synthetic Biology. In this work, the genetic elements required for CRISPRs-based editing and transcriptional regulation were adapted to GB, and a workflow for gRNAs construction was designed and optimized. New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site.

Conclusions

The functionality and the efficiency of gRNA–Cas9 GB tools were demonstrated in Nicotiana benthamiana using transient expression assays both for gene targeted mutations and for transcriptional regulation. The availability of gRNA–Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineering.

High-frequency, precise modification of the tomato genome (open access)

Čermák, T., Baltes, N. J., Čegan, R., Zhang, Y., & Voytas, D. F. (2015). High-frequency, precise modification of the tomato genome. Genome biology, 16(1), 1-15. doi:10.1186/s13059-015-0796-9 The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.

Plant synthetic promoters and transcription factors

Liu, W., & Stewart, C. N. (2016). Plant synthetic promoters and transcription factors. Current opinion in biotechnology, 37, 36-44. doi:10.1016/j.copbio.2015.10.001 Synthetic promoters and transcription factors (TFs) have become incredibly powerful and efficient components for precise regulation of targeted plant transgene expression. Synthetic promoters can be rationally designed and constructed using specific type, copy number and spacing of motifs placed upstream of synthetic or native core promoters. Similarly, synthetic TFs can be constructed using a variety of DNA binding domains (DBDs) and effector domains. Synthetic promoters and TFs can provide tremendous advantages over their natural counterparts with regards to transgene expression strength and specificity. They will probably be needed for coordinated transgene expression for metabolic engineering and synthetic circuit applications in plants for bioenergy and advanced crop engineering. In this article we review the recent advances in synthetic promoters and TFs in plants and speculate on their future.