Cloning of the rice Xo1 resistance gene and interaction of the Xo1 protein with the defense-suppressing Xanthomonas effector Tal2h

The Xo1 locus in the heirloom rice variety Carolina Gold Select confers resistance to bacterial leaf streak and bacterial blight, caused by Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae, respectively. Resistance is triggered by pathogen-delivered transcription activator-like effectors (TALEs) independent of their ability to activate transcription and is suppressed by truncated variants called truncTALEs, common among Asian strains. By transformation of the susceptible variety Nipponbare, we show that one of 14 nucleotide-binding, leucine-rich repeat (NLR) protein genes at the locus, with a zinc finger BED domain, is the Xo1 gene. Analyses of published transcriptomes revealed that the Xo1-mediated response is more similar to those mediated by two other NLR resistance genes than it is to the response associated with TALE-specific transcriptional activation of the executor resistance gene Xa23 and that a truncTALE dampens or abolishes activation of defense-associated genes by Xo1. In Nicotiana benthamiana leaves, fluorescently tagged Xo1 protein, like TALEs and truncTALEs, localized to the nucleus. And endogenous Xo1 specifically coimmunoprecipitated from rice leaves with a pathogen-delivered, epitope-tagged truncTALE. These observations suggest that suppression of Xo1-function by truncTALEs occurs through direct or indirect physical interaction. They further suggest that effector coimmunoprecipitation may be effective for identifying or characterizing other resistance genes

Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online

TALEN-mediated editing of the mouse Y chromosome

The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes—Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.National Institutes of Health (U.S.) (US NIH grant R01-HG000257)National Institutes of Health (U.S.) (US NIH grant R01-CA084198)National Institutes of Health (U.S.) (US NIH grant R37-HD045022)Croucher Foundation (Scholarship)Howard Hughes Medical Institute (Investigator

Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a ‘modular assembly’ method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry

Efficient TALEN-mediated gene knockout in livestock

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications

DNA sense-and-respond protein modules for mammalian cells

We generated synthetic protein components that can detect specific DNA sequences and subsequently trigger a desired intracellular response. These modular sensors exploit the programmability of zinc-finger DNA recognition to drive the intein-mediated splicing of an artificial trans-activator that signals to a genetic circuit containing a given reporter or response gene. We used the sensors to mediate sequence recognition−induced apoptosis as well as to detect and report a viral infection. This work establishes a synthetic biology framework for endowing mammalian cells with sentinel capabilities, which provides a programmable means to cull infected cells. It may also be used to identify positively transduced or transfected cells, isolate recipients of intentional genomic edits and increase the repertoire of inducible parts in synthetic biology.United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-23)Defense Threat Reduction Agency (DTRA) (HDTRA1-14-1-0006)United States. Air Force Office of Scientific Research (FA9550-14-1-0060

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

[EN] 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 con¿ struction 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 engineeringThis work has been funded by Grant BIO2013-42193-R from Plan Nacional I + D of the Spanish Ministry of Economy and Competitiveness. Vazquez-Vilar M. is a recipient of a Junta de Ampliacion de Estudios fellowship. Bernabe-Orts J.M. is a recipient of a FPI fellowship. We want to thank Nicola J. Patron and Mark Youles for kindly providing humanCas9 and U6-26 clones. We also want to thank Eugenio Gomez for providing Arabidopsis thaliana genomic DNA and Concha Domingo for providing rice genomic DNA. We also want to thank the COST Action FA1006 for the support in the development of the software tools.Vázquez-Vilar, M.; Bernabé-Orts, JM.; Fernández Del Carmen, MA.; Ziarsolo Areitioaurtena, P.; Blanca Postigo, JM.; Granell Richart, A.; Orzáez Calatayud, DV. (2016). A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods. 12. https://doi.org/10.1186/s13007-016-0101-2S12Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308. doi: 10.1038/nprot.2013.143 .Yang X. Applications of CRISPR-Cas9 mediated genome engineering. Mil Med Res. 2015;2:11. doi: 10.1186/s40779-015-0038-1 .Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. 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The Tomato Sequencing Project, the First Cornerstone of the International Solanaceae Project (SOL)

The genome of tomato (Solanum lycopersicum) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, The Netherlands, France, Japan, Spain, Italy and the United States) as part of a larger initiative called the ‘International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation’. The goal of this grassroots initiative, launched in November 2003, is to establish a network of information, resources and scientists to ultimately tackle two of the most significant questions in plant biology and agriculture: (1) How can a common set of genes/proteins give rise to a wide range of morphologically and ecologically distinct organisms that occupy our planet? (2) How can a deeper understanding of the genetic basis of plant diversity be harnessed to better meet the needs of society in an environmentally friendly and sustainable manner? The Solanaceae and closely related species such as coffee, which are included in the scope of the SOL project, are ideally suited to address both of these questions. The first step of the SOL project is to use an ordered BAC approach to generate a high quality sequence for the euchromatic portions of the tomato as a reference for the Solanaceae. Due to the high level of macro and micro-synteny in the Solanaceae the BAC-by-BAC tomato sequence will form the framework for shotgun sequencing of other species. The starting point for sequencing the genome is BACs anchored to the genetic map by overgo hybridization and AFLP technology. The overgos are derived from approximately 1500 markers from the tomato high density F2-2000 genetic map (http://sgn.cornell.edu/). These seed BACs will be used as anchors from which to radiate the tiling path using BAC end sequence data. Annotation will be performed according to SOL project guidelines. All the information generated under the SOL umbrella will be made available in a comprehensive website. The information will be interlinked with the ultimate goal that the comparative biology of the Solanaceae—and beyond—achieves a context that will facilitate a systems biology approach