Genetic dissection of mutagenic repair and T-DNA capture at CRISPR-induced DNA breaks in .

A practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via transformation. The double-strand break (DSB)-inducing enzyme is expressed from a transferred segment of bacterial DNA, the T-DNA, which upon transformation integrates at random locations into the host genome or is captured at the self-inflicted DSB site. To develop efficient strategies for precise genome editing, it is thus important to define the mechanisms that repair CRISPR-induced DSBs, as well as those that govern random and targeted integration of T-DNA.

Distinct mechanisms for genomic attachment of the 5' and 3' ends of Agrobacterium T-DNA in plants.

Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides the T-DNA into the plant's nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown.

Gene targeting in polymerase theta-deficient Arabidopsis thaliana.

Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events.

CRISPR/Cas9 Mutagenesis by Translocation of Cas9 Protein Into Plant Cells via the Type IV Secretion System.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool for genome engineering in plants. The RNA-guided Cas9 endonuclease is usually delivered into plant cells as a DNA construct encoding Cas9 and the single guide RNA (sgRNA). However, constitutive expression of nucleases may cause off target mutations.

True gene-targeting events by CRISPR/Cas-induced DSB repair of the PPO locus with an ectopically integrated repair template.

In recent years, several tools have become available for improved gene-targeting (GT) in plants. DNA breaks at specific sites activate local DNA repair and recombination, including recombination with ectopic sequences leading to GT. Large-scale transformation with the repair template can be avoided by pre-insertion of the repair template in the genome and liberation by sequence-specific nucleases (in planta GT procedure).

CRISPR/Cas9-Induced Double-Strand Break Repair in Arabidopsis Nonhomologous End-Joining Mutants.

Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs).

T-DNA integration in plants results from polymerase-θ-mediated DNA repair.

Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops.

Epigenetic alterations at genomic loci modified by gene targeting in Arabidopsis thaliana.

Gene Targeting (GT) is the integration of an introduced vector into a specific chromosomal site, via homologous recombination. It is considered an effective tool for precise genome editing, with far-reaching implications in biological research and biotechnology, and is widely used in mice, with the potential of becoming routine in many species. Nevertheless, the epigenetic status of the targeted allele remains largely unexplored.

DAYSLEEPER: a nuclear and vesicular-localized protein that is expressed in proliferating tissues.

Background: DAYSLEEPER is a domesticated transposase that is essential for development in Arabidopsis thaliana [Nature, 436:282-284, 2005]. It is derived from a hAT-superfamily transposon and contains many of the features found in the coding sequence of these elements [Nature, 436:282-284, 2005, Genetics, 158:949-957, 2001]. This work sheds light on the expression of this gene and localization of its product in protoplasts and in planta.

Poly(ADP-ribose)polymerases are involved in microhomology mediated back-up non-homologous end joining in Arabidopsis thaliana.

Besides the KU-dependent classical non-homologous end-joining (C-NHEJ) pathway, an alternative NHEJ pathway first identified in mammalian systems, which is often called the back-up NHEJ (B-NHEJ) pathway, was also found in plants. In mammalian systems PARP was found to be one of the essential components in B-NHEJ. Here we investigated whether PARP1 and PARP2 were also involved in B-NHEJ in Arabidopsis.

ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation.

Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5' coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events.

The SLEEPER genes: a transposase-derived angiosperm-specific gene family.

Background: DAYSLEEPER encodes a domesticated transposase from the hAT-superfamily, which is essential for development in Arabidopsis thaliana. Little is known about the presence of DAYSLEEPER orthologs in other species, or how and when it was domesticated. We studied the presence of DAYSLEEPER orthologs in plants and propose a model for the domestication of the ancestral DAYSLEEPER gene in angiosperms.

Transposon proliferation in an asexual parasitoid.

The widespread occurrence of sex is one of the most elusive problems in evolutionary biology. Theory predicts that asexual lineages can be driven to extinction by uncontrolled proliferation of vertically transmitted transposable elements (TEs), which accumulate because of the inefficiency of purifying selection in the absence of sex and recombination. To test this prediction, we compared genome-wide TE load between a sexual lineage of the parasitoid wasp Leptopilina clavipes and a lineage of the same species that is rendered asexual by Wolbachia-induced parthenogenesis.

Programmed Cell Death in the Leaves of the Arabidopsis Spontaneous Necrotic Spots (sns-D) Mutant Correlates with Increased Expression of the Eukaryotic Translation Initiation Factor eIF4B2.

From a pool of transgenic Arabidopsis (Arabidopsis thaliana) plants harboring an activator T-DNA construct, one mutant was identified that developed spontaneous necrotic spots (sns-D) on the rosette leaves under aseptic conditions. The sns-D mutation is dominant and homozygous plants are embryo lethal. The mutant produced smaller rosettes with a different number of stomata than the wild-type.

ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double-strand breaks (DSBs) at a specific locus. These DSBs may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway that is used. These promising techniques for genome engineering were evaluated in Arabidopsis plants using Agrobacterium-mediated floral dip transformation.

Responsiveness of exercise parameters in children with inflammatory myositis.

Objective: Juvenile dermatomyositis (DM) is an inflammatory myopathy in which the immune system targets the microvasculature of the skeletal muscle and skin, leading to significant muscle weakness and exercise intolerance, although the precise etiology is unknown. The goal of this study was to investigate the changes in exercise capacity in children with myositis during active and inactive disease periods and to study the responsiveness of exercise parameters.

Methods: Thirteen children with juvenile DM (mean+/-SD age 11.

Manipulation of starch granule size distribution in potato tubers by modulation of plastid division.

Starch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery are likely to be similar for all plastids.

Glucosylation activity and complex formation of two classes of reversibly glycosylated polypeptides.

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch. We have isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function of RGPs.

Macrophage/microglial cell subpopulations in glioblastoma multiforme relapses are differentially altered by radiochemotherapy.

Following surgical removal of glioblastoma multiforme (GBM), radiochemotherapy impedes neoplastic outgrowth and relapse formation. Macrophages/microglial cells are believed to be potent mediators of the host defense system in GBM. However, little is known about their alteration by postsurgical therapies.

B-type granule containing protrusions and interconnections between amyloplasts in developing wheat endosperm revealed by transmission electron microscopy and GFP expression.

Starch granules in mature wheat endosperm show a bimodal size distribution. The formation of small starch granules in wheat endosperm cells was studied by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) after expression and targeting of fluorescent protein into amyloplasts. Both techniques demonstrated the presence of protrusions emanating from A-type granules-containing amyloplasts and the presence of B-type starch granules in these evaginations.

RAP-1 is an Arabidopsis MYC-like R protein homologue, that binds to G-box sequence motifs.

An Arabidopsis cDNA clone encoding a DNA-binding protein, RAP-1, was isolated by southwestern screening of an Escherichia coli cDNA expression library. The protein contains a bHLH DNA-binding domain and is homologous to R proteins, regulating anthocyanin biosynthesis. RAP-1 binds to the sequence CACNTG.

Characterization of a zinc-dependent transcriptional activator from Arabidopsis.

The C2-H2 zinc-finger is a widely occurring DNA binding motif, usually present as tandem repeats. The majority of C2-H2 zinc-finger proteins that have been studied are derived from animals. Here, we characterize a member of a distinct class of plant C2-H2 zinc-finger proteins in detail.

The 22 bp W1 element in the pea lectin promoter is necessary and, as a multimer, sufficient for high gene expression in tobacco seeds.

The pea lectin (Psl) gene encodes an abundant seed protein. Its seed-specific expression pattern is conserved in transgenic tobacco plants. Progressive 5' promoter deletions resulted in a gradual decrease of transcriptional activity in tobacco seed.