Abscisic acid (ABA) regulates numerous developmental processes and adaptive stress responses in plants. Many ABA signaling components have been identified, but their interconnections and a consensus on the structure of the ABA signaling network have eluded researchers. Recently, several advances have led to the identification of ABA receptors and their three-dimensional structures, and an understanding of how key regulatory phosphatase and kinase activities are controlled by ABA. A new model for ABA action has been proposed and validated, in which the soluble PYR/PYL/RCAR receptors function at the apex of a negative regulatory pathway to directly regulate PP2C phosphatases, which in turn directly regulate SnRK2 kinases. This model unifies many previously defined signaling components and highlights the importance of future work focused on defining the direct targets of SnRK2s and PP2Cs, dissecting the mechanisms of hormone interactions (i.e., cross talk) and defining connections between this new negative regulatory pathway and other factors implicated in ABA signaling.

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Abscisic Acid: Emergence of a Core Signaling Network

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

Plants respond to survive under water-deficit conditions via a series of physiological, cellular, and molecular processes culminating in stress tolerance. Many drought-inducible genes with various functions have been identified by molecular and genomic analyses in Arabidopsis, rice, and other plants, including a number of transcription factors that regulate stress-inducible gene expression. The products of stress-inducible genes function both in the initial stress response and in establishing plant stress tolerance. In this short review, recent progress resulting from analysis of gene expression during the drought-stress response in plants as well as in elucidating the functions of genes implicated in the stress response and/or stress tolerance are summarized. A description is also provided of how various genes involved in stress tolerance were applied in genetic engineering of dehydration stress tolerance in transgenic Arabidopsis plants.

Gene networks involved in drought stress response and tolerance

Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells - this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.

/pdf/genome-editing-with-engineered-zinc-finger-nucleases-3i1sf7uaja.pdf

Genome editing with engineered zinc finger nucleases

Plants respond to survive under water-deficit conditions via a series of physiological, cellular, and molecular processes culminating in stress tolerance. Many drought-inducible genes with various functions have been identified by molecular and genomic analyses in Arabidopsis, rice, and other plants, including a number of transcription factors that regulate stress-inducible gene expression. The products of stress-inducible genes function both in the initial stress response and in establishing plant stress tolerance. In this short review, recent progress resulting from analysis of gene expression during the drought-stress response in plants as well as in elucidating the functions of genes implicated in the stress response and/or stress toler ance are summarized. A description is also provided of how various genes involved in stress tolerance were applied in genetic engineering of dehydration stress tolerance in transgenic Arabidopsis plants.

Gene networks involved in drought stress response and

Genome editing can be achieved by inducing site-directed genome modification using artificial nucleases. The CRISPR/Cas9 system employs an RNA-guided nuclease that specifically recognizes and digests the target DNA of interest and has been utilized widely in genome editing in various organisms, including crops. Attempts have been made to optimize and improve CRISPR/Cas9 technology specifically for crop genome editing. Furthermore, new methods for introducing CRISPR/Cas9 tools into the cells of crop plants are also now being developed. CRISPR/Cas9 technology not only is a basic research tool but also has become one of the most useful molecular breeding tools currently available, and superior traits in various crop species have now been established. In this chapter, we describe the current status and perspectives of crop molecular breeding using CRISPR/Cas9.

Crop Breeding Using CRISPR/Cas9

RPK2 is a novel factor controlling anther development in Arabidopsis thaliana

SUMMARY Adoption of the CRISPR-Cas system has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—has been less developed. Type I systems in which Cas3 nuclease degrades the target DNA are known; in contrast, for the sub-type CRISPR type I-D (TiD), which lacks a typical Cas3 nuclease in its cascade, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d—a nuclease in TiD—is multi-functional in PAM recognition, stabilization and target DNA degradation. TiD can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. TiD off-target effects, which were dependent on the mismatch position in the protospacer of TiD, were also identified. Our findings suggest TiD as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

/pdf/genome-editing-in-mammals-and-plants-using-crispr-type-i-d-2p6utyqesj.pdf

Genome editing in mammals and plants using CRISPR type I-D nuclease


 Background
Genome editing technique is a powerful tool in plant genetic engineering to accelerate the rapid breeding of crop plants. Currently, obtaining transgene-free mutant plants is an important issue. transgene-free mutant plants such as null-segregant, can be segregated out from selfing or back-crossed populations. However, such procedures are time consuming, and, therefore, the development of efficient systems to eliminate transgenes in mutant plants is required.
Results
CRISPR/Cas9 vectors targeting Sl IAA9 were introduced into the tomato cultivars, Ailsa Craig, Moneymaker, Super Roma and Rio Grande, via Agrobacterium transformation. Bi-allelic mutations were detected at ratios of 9–46% in CRISPR/Cas9-transgenic T0 shoots. Sequence analysis revealed that the bi-allelic mutations generated stop codons by frame-shift of SlIAA9 amino acid sequences, indicating that sliaa9 knockout mutants with parthenocarpic phenotypes were generated in the T0 generation with high efficiency in these cultivars. Subsequently, many null-segregant (transgene free) lines, as confirmed by PCR and Southern blot, were isolated rapidly in the T1 generation by self-pollination.
Conclusions
The method used in this study is effective in achieving rapid isolation of null-segregants with high efficiency, while adding important traits to various cultivars at the same time. Our developed system would further increase the advantages of using CRISPR/Cas9 for the production of novel crop varieties in the future.

/pdf/efficient-generation-of-null-segregant-parthenocarpic-tomato-22qsutz9c2.pdf

Efficient generation of null-segregant parthenocarpic tomato by CRISPR/Cas9 editing

Genome editing with artificially engineered nucleases is an advanced molecular technology for pursuing precise and effective genetic engineering. In this technology, engineered nucleases induce DNA double-strand breaks at targeted sites in a genome, stimulating the DNA repair system in cells, thus enabling site-directed mutagenesis. Genome editing using CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein9 (Cas9), originating from a defence system of prokaryotes, is a powerful technology that is now being widely utilized in molecular research studies, as well as in breeding programmes of various plant species, including fruit trees, to impart either novel or enhanced traits to established commercial cultivars or to new cultivars/genotypes. Recently, several reports have demonstrated successful apple genome editing and the introduction of important traits, such as those for early flowering and reduced fire blight susceptibility, to popular commercial cultivars, such as ‘Gala’ and ‘Golden Delicious’. It is important to point out that these reports reveal that such genome-edited/mutant apple plants or cell lines do not carry foreign genes. Nevertheless, during the process of precise genome editing, the coexistence of various types of mutations referred to as “mosaic mutations” and off-target effects are major concerns. Therefore, to minimize such effects, selection of target sequences and estimation of off-target effects for CRISPR/Cas9 has been developed for many organisms, and these have also been employed for apple by using in silico analysis based on genome information. On the other hand, apple genome heterozygosity has led to difficulties in genome editing, as the complex genome of apple precludes the use of some of these basic techniques for genome editing. Therefore, further studies focused on genome information and culture techniques tailored for apple are needed. It will be highly critical for each apple cultivar in developing precise and efficient genome editing for apple. This chapter will provide an overview of current studies of genome editing in apple and will discern and explore how this strategy will provide insights into molecular breeding technologies for genetic improvement of the apple.

Yuriko Osakabe

Papers

Crop Breeding Using CRISPR/Cas9

RPK2 is a novel factor controlling anther development in Arabidopsis thaliana

Genome editing in mammals and plants using CRISPR type I-D nuclease

Efficient generation of null-segregant parthenocarpic tomato by CRISPR/Cas9 editing

Genome Editing in Apple