Revolutionizing plant biology: Multiple ways of genome engineering by CRISPR/Cas

The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by site-specific nucleases to initiate DNA repair reactions that are either based on non-homologous end joining (NHEJ) or homologous recombination (HR). Recently, the CRISPR/Cas system emerged as the most important tool for genome engineering due to its simple structure and its applicability to a wide range of organisms. Here, we review the current status of its various applications in plants, where it is used for the successful generation of stable mutations in a steadily growing number of species through NHEJ. Furthermore, tremendous progress in plant genome engineering by HR was obtained by the setup of replicon mediated and in planta gene targeting techniques. Finally, other complex approaches that rely on the induction of more than one DNA lesion at a time such as paired nickases to avoid off-site effects or controlled genomic deletions are beginning to be applied routinely

Untersuchungen zur Gentechnologie und DNA-Reparatur in Pflanzen mithilfe der Cas9 Nickase

Ausgehend vom bakteriellen CRISPR/Cas System wurde ein Expressionsystem für Cas9 Nukleasen und Nickasen in Pflanzen etabliert. Deep Sequencing und Quantifizierung homologer Rekombination zeigten hocheffiziente Induktion von Einzel- und Doppelstrangbrüchen. Darüber hinaus wurden gepaarte Cas9 Nickasen zur gezielten, stabilen Mutagenese sowie zur Analyse komplexer Genomveränderungen eingesetzt

Early Attachment Disruption, Inflammation, and Vulnerability for Depression in Rodent and Primate Models

Early experiments in nonhuman primates established the relation between disruption of filial attachment and depressive-like outcomes. Subsequent studies in rats and mice have been instrumental in linking depressive-like outcomes to disturbances in maternal behavior. Another aspect of attachment disruption, absence of the attachment object per se, may be studied more effectively in a different laboratory rodent—the guinea pig. Here, we discuss the rationale for using guinea pigs for this work. We then review guinea pig studies providing evidence for inflammatory mechanisms mediating both depressive-like behavior during separation as well as sensitization of stress responsiveness such as is thought to lead to increased vulnerability to depression at later ages. Finally, we discuss recent complementary work in adult monkeys that suggests cross-species generalizability of broad principles derived from the guinea pig experiments. Overall, the findings provide experimental support for human research implicating inflammatory mechanisms in the development of increased stress responsiveness and vulnerability to depression following attachment disruption and other forms of early-life stress. Specifically, the findings suggest inflammatory mechanisms may set in motion a cascade of underlying processes that mediate later increased stress responsiveness and, therefore, depression susceptibility

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

Currently, biology is revolutionized by ever growing applications of the CRISPR /Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence‐specific DN ases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RN ase Cas13 (formerly named C2c2). Although double strand break‐induced gene editing based on error‐prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR /Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9‐ and Cas12‐ mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence‐based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNA s and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNA s and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way

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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Plant phenomics, from sensors to knowledge

Major improvements in crop yield are needed to keep pace with population growth and climate change. While plant breeding efforts have greatly benefited from advances in genomics, profiling the crop phenome (i.e., the structure and function of plants) associated with allelic variants and environments remains a major technical bottleneck. Here, we review the conceptual and technical challenges facing plant phenomics. We first discuss how, given plants’ high levels of morphological plasticity, crop phenomics presents distinct challenges compared with studies in animals. Next, we present strategies for multi-scale phenomics, and describe how major improvements in imaging, sensor technologies and data analysis are now making high-throughput root, shoot, whole-plant and canopy phenomic studies possible. We then suggest that research in this area is entering a new stage of development, in which phenomic pipelines can help researchers transform large numbers of images and sensor data into knowledge, necessitating novel methods of data handling and modelling. Collectively, these innovations are helping accelerate the selection of the next generation of crops more sustainable and resilient to climate change, and whose benefits promise to scale from physiology to breeding and to deliver real world impact for ongoing global food security efforts

The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency

Androgen and glucocorticoid levels reflect seasonally occurring social challenges in male redfronted lemurs (Eulemur fulvus rufus)

Intense reproductive competition and social instability are assumed to increase concentrations of glucocorticoids and androgens in vertebrates, as a means of coping with these challenges. In seasonally breeding redfronted lemurs (Eulemur fulvus rufus), the mating and the birth season and the associated increased male competition are predicted to pose such reproductive challenges. In this paper, we investigate seasonal variation in hormone excretion in male redfronted lemurs, and examine whether this variation is associated with social or ecological factors. Although dominance status has been shown to affect individual stress levels across many taxa, we predicted no rank-related differences in glucocorticoids for redfronted lemurs because relatively equal costs are associated with both high and low rank positions (based on patterns of rank acquisition/maintenance and threats toward subordinates). Over a 14-month period, we collected behavioral data (1843 focal hours) and 617 fecal samples from 13 redfronted lemur males in Kirindy Forest/Madagascar. We found no general rank-related pattern of testosterone or glucocorticoid excretion in this species. Both hormones were excreted at significantly higher levels during the mating and the birth season, despite social stability during both periods. The elevated mating season levels may be explained by increased within-group reproductive competition during this time and are in line with previous studies of other seasonally reproducing primates. For the birth season increase, we propose that the predictable risk of infanticide in this highly seasonal species affects male gonadal and adrenal endocrine activity. We evaluate alternative social and ecological factors influencing the production of both hormone classes and conclude based on our preliminary investigations that none of them can account for the observed pattern

Visual Information Alone Changes Behavior and Physiology during Social Interactions in a Cichlid Fish (Astatotilapia burtoni)

Social behavior can influence physiological systems dramatically yet the sensory cues responsible are not well understood. Behavior of male African cichlid fish, Astatotilapia burtoni, in their natural habitat suggests that visual cues from conspecifics contribute significantly to regulation of social behavior. Using a novel paradigm, we asked whether visual cues alone from a larger conspecific male could influence behavior, reproductive physiology and the physiological stress response of a smaller male. Here we show that just seeing a larger, threatening male through a clear barrier can suppress dominant behavior of a smaller male for up to 7 days. Smaller dominant males being “attacked” visually by larger dominant males through a clear barrier also showed physiological changes for up to 3 days, including up-regulation of reproductive- and stress-related gene expression levels and lowered plasma 11-ketotestesterone concentrations as compared to control animals. The smaller males modified their appearance to match that of non-dominant males when exposed to a larger male but they maintained a physiological phenotype similar to that of a dominant male. After 7 days, reproductive- and stress- related gene expression, circulating hormone levels, and gonad size in the smaller males showed no difference from the control group suggesting that the smaller male habituated to the visual intruder. However, the smaller male continued to display subordinate behaviors and assumed the appearance of a subordinate male for a full week despite his dominant male physiology. These data suggest that seeing a larger male alone can regulate the behavior of a smaller male but that ongoing reproductive inhibition depends on additional sensory cues. Perhaps, while experiencing visual social stressors, the smaller male uses an opportunistic strategy, acting like a subordinate male while maintaining the physiology of a dominant male