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#SRDX DNA RECOGNITION FULL#
To exploit the full potential of the T-DNA strategy it is important to expand the ability to combine different gRNAs together with Cas9 within a single T-DNA, as it has been demonstrated that all-in-one plasmid approaches significantly increase editing efficiency. In this case, T-DNA-delivered gRNA–Cas9, besides acting transiently during callus formation, can also integrate in the genome and continue its activity in somatic tissues. A successful alternative for plants is the use of Agrobacterium mediated T-DNA transformation, followed by callus induction and organogenic plant regeneration (or floral dip transformation in the case of Arabidopsis). The direct transfection of Cas9 and guide RNAs into plant protoplasts followed by plant regeneration from single-cell has been shown effective for genome editing in rice and tobacco, however the efficiency remained relatively low, and besides, whole plant regeneration from protoplasts is not currently feasible for many crop species. While similar technologies such as the ZFNs (zinc finger nucleases) or the TAL effectors require recoding of a new protein for each target sequence, with the gRNA–Cas9 a change of 20 nts in the guide RNA is enough, paving the way for multiplex editing and design of complex regulatory circuits among other engineering possibilities. A remarkable feature of gRNA–Cas9 is that facilitates targeting multiple sequences simultaneously. On the plant field, RNA-guided genome engineering via Cas9 has been employed in diverse approaches, from single and/or multiple gene knock-outs to targeted insertions of donor sequences or even targeted transcriptional regulation through the fusion of transcriptional activation or repressor domains to an inactivated Cas9. The application of the RNA-guided Cas9 technology is being widely exploited by the scientific community in cell cultures, animals or plants. This system is based on a guide RNA (gRNA) that directs the Streptococcus pyogenes Cas9 nuclease to its target site. Since its discovery, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune bacterial system has rapidly become a powerful technology for genome editing in many organisms. The availability of gRNA–Cas9 GB toolbox will facilitate the application of CRISPR/Cas9 technology to plant genome engineering. 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.
#SRDX DNA RECOGNITION SOFTWARE#
New software tools specific for CRISPRs assembly were created and incorporated to the public GB resources site. 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. 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. 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. 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 efficiency, versatility and multiplexing capacity