Genome editing in wheat microspores and haploid embryos mediated by delivery of ZFN proteins and cell-penetrating peptide complexes.

Recent advances in genome engineering technologies based on designed endonucleases (DE) allow specific and predictable alterations in plant genomes to generate value-added traits in crops of choice. The EXZACT Precision technology, based on zinc finger nucleases (ZFN), has been successfully used in the past for introduction of precise mutations and transgenes to generate novel and desired phenotypes in several crop species. Current methods for delivering ZFNs into plant cells are based on traditional genetic transformation methods that result in stable integration of the nuclease in the genome. Here, we describe for the first time, an alternative ZFN delivery method where plant cells are transfected with ZFN protein that eliminates the need for stable nuclease genomic integration and allows generation of edited, but not transgenic cells or tissues. For this study, we designed ZFNs targeting the wheat IPK1 locus, purified active ZFN protein from bacterial cultures, complexed with cell-penetrating peptides (CPP) and directly transfected the complex into either wheat microspores or embryos. NGS analysis of ZFN-treated material showed targeted edits at the IPK1 locus in independent experiments. This is the first description of plant microspore genome editing by a ZFN when delivered as a protein complexed with CPP.

Short-term toxicogenomics as an alternative approach to chronic in vivo studies for derivation of points of departure: A case study in the rat with a triazole fungicide.

The derivation of an apical endpoint point of departure (POD) from animal-intensive testing programs has been the traditional cornerstone of human health risk assessment. Replacement of in vivo chronic studies with novel approaches, such as toxicogenomics, holds promise for future alternative testing paradigms that significantly reduce animal testing. We hypothesized that a toxicogenomic POD following a 14 day exposure in the rat would approximate the most sensitive apical endpoint POD derived from a battery of chronic, carcinogenicity, reproduction and endocrine guideline toxicity studies. To test this hypothesis, we utilized myclobutanil, a triazole fungicide, as a model compound. In the 14 day study, male rats were administered 0 (vehicle), 30, 150, or 400 mg/kg/day myclobutanil via oral gavage. Endpoints evaluated included traditional apical, hormone, and liver and testis transcriptomic (whole genome RNA sequencing) data. From the transcriptomic data, liver and testis biological effect POD (BEPOD) values were derived. Myclobutanil exposure for 14 days resulted in increased liver weight, altered serum hormones, liver histopathology, and differential gene expression in liver and testis. The liver and testis BEPODs from the short-term study were 22.2 and 25.4 mg/kg/day, respectively. These BEPODs were approximately an order of magnitude higher than the most sensitive apical POD identified from the two year cancer bioassay based on testis atrophy (1.4 mg/kg/day). This study demonstrates the promise of using a short-term study BEPOD to derive a POD for human health risk assessment while substantially reducing animal testing.

Integration of omics approaches to understand oil/protein content during seed development in oilseed crops.

Oilseed crops, especially soybean (Glycine max) and canola/rapeseed (Brassica napus), produce seeds that are rich in both proteins and oils and that are major sources of energy and nutrition worldwide. Most of the nutritional content in the seed is accumulated in the embryo during the seed filling stages of seed development. Understanding the metabolic pathways that are active during seed filling and how they are regulated are essential prerequisites to crop improvement. In this review, we summarize various omics studies of soybean and canola/rapeseed during seed filling, with emphasis on oil and protein traits, to gain a systems-level understanding of seed development. Currently, most (80-85%) of the soybean and rapeseed reference genomes have been sequenced (950 and 850 megabases, respectively). Parallel to these efforts, extensive omics datasets from different seed filling stages have become available. Transcriptome and proteome studies have detected preponderance of starch metabolism and glycolysis enzymes to be the possible cause of higher oil in B. napus compared to other crops. Small RNAome studies performed during the seed filling stages have revealed miRNA-mediated regulation of transcription factors, with the suggestion that this interaction could be responsible for transitioning the seeds from embryogenesis to maturation. In addition, progress made in dissecting the regulation of de novo fatty acid synthesis and protein storage pathways is described. Advances in high-throughput omics and comprehensive tissue-specific analyses make this an exciting time to attempt knowledge-driven investigation of complex regulatory pathways.

A quantitative atlas of polyadenylation in five mammals.

We developed PolyA-seq, a strand-specific and quantitative method for high-throughput sequencing of 3' ends of polyadenylated transcripts, and used it to globally map polyadenylation (polyA) sites in 24 matched tissues in human, rhesus, dog, mouse, and rat. We show that PolyA-seq is as accurate as existing RNA sequencing (RNA-seq) approaches for digital gene expression (DGE), enabling simultaneous mapping of polyA sites and quantitative measurement of their usage. In human, we confirmed 158,533 known sites and discovered 280,857 novel sites (FDR < 2.5%). On average 10% of novel human sites were also detected in matched tissues in other species. Most novel sites represent uncharacterized alternative polyA events and extensions of known transcripts in human and mouse, but primarily delineate novel transcripts in the other three species. A total of 69.1% of known human genes that we detected have multiple polyA sites in their 3'UTRs, with 49.3% having three or more. We also detected polyadenylation of noncoding and antisense transcripts, including constitutive and tissue-specific primary microRNAs. The canonical polyA signal was strongly enriched and positionally conserved in all species. In general, usage of polyA sites is more similar within the same tissues across different species than within a species. These quantitative maps of polyA usage in evolutionarily and functionally related samples constitute a resource for understanding the regulatory mechanisms underlying alternative polyadenylation.

Trait stacking via targeted genome editing.

Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.

A microRNA or messenger RNA point of departure estimates an apical endpoint point of departure in a rat developmental toxicity model.

Traditional developmental toxicity testing practice examines fetal apical endpoints to identify a point of departure (POD) for risk assessment. A potential new testing paradigm involves deriving a POD from a comprehensive analysis of molecular-level change. Here, the rat ketoconazole endocrine-mediated developmental toxicity model was used to test the hypothesis that maternal epigenomic (miRNA) and transcriptomic (mRNA) PODs are similar to fetal apical endpoint PODs. Sprague-Dawley rats were exposed from gestation day (GD) 6-21 to 0, 0.063, 0.2, 0.63, 2, 6.3, 20, or 40 mg/kg/day ketoconazole. Dam systemic, liver, and placenta PODs, along with GD 21 fetal resorption, body weight, and skeletal apical PODs were derived using BMDS software. GD 21 dam liver and placenta miRNA and mRNA PODs were obtained using three methods: a novel individual molecule POD accumulation method, a first mode method, and a gene set method. Dam apical POD values ranged from 2.0 to 38.6 mg/kg/day; the lowest value was for placenta histopathology. Fetal apical POD values were 10.9-20.3 mg/kg/day; the lowest value was for fetal resorption. Dam liver miRNA and mRNA POD values were 0.34-0.69 mg/kg/day, and placenta miRNA and mRNA POD values were 2.53-6.83 mg/kg/day. Epigenomic and transcriptomic POD values were similar across liver and placenta. Deriving a molecular POD from dam liver or placenta was protective of a fetal apical POD. These data support the conclusion that a molecular POD can be used to estimate, or be protective of, a developmental toxicity apical POD.

Dioxin male rat reproductive toxicity mode of action and relative potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran characterized by fetal pituitary and testis transcriptome profiling.

Fetal rat exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces epididymal sperm number involving altered pituitary-testicular hormonal signaling as the proposed mode-of-action (MOA). To evaluate this MOA and compare TCDD to 2,3,7,8-tetrachlorodibenzofuran (TCDF), an in utero rat exposure and study was conducted. Endpoints included congener tissue levels and transcriptomes of maternal liver and fetal liver, testis, and pituitary. Decreased gonadotropin subunit mRNAs levels (Lhb and Fshb) and enriched signaling pathways including GNRH Signaling and Calcium Signaling were observed in fetal pituitary after TCDD (but not TCDF) exposure. TCDD (but not TCDF) decreased fetal testis cholesterologenic and steroidogenic pathway genes. TCDD tissue concentrations in dam liver, dam adipose, and whole fetus were approximately 3- to 6-fold higher than TCDF. These results support a MOA for dioxin-induced rat male reproductive toxicity involving key events in both the fetal pituitary (e.g., reduced gonadotropin production) and fetal testis (e.g., reduced Leydig cell cholesterologenesis and steroidogenesis).

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.