The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli

Genome engineering methods in E. coli allow for easy to perform manipulations of the chromosome in vivo with the assistance of the λ-Red recombinase system. These methods generally rely on the insertion of an antibiotic resistance cassette followed by removal of the same cassette, resulting in a two-step procedure for genomic manipulations. Here we describe a method and plasmid system that can edit the genome of E. coli without chromosomal markers. This system, known as Scarless Cas9 Assisted Recombineering (no-SCAR), uses λ-Red to facilitate genomic integration of donor DNA and double stranded DNA cleavage by Cas9 to counterselect against wild-type cells. We show that point mutations, gene deletions, and short sequence insertions were efficiently performed in several genomic loci in a single-step with regards to the chromosome and did not leave behind scar sites. The single-guide RNA encoding plasmid can be easily cured due to its temperature sensitive origin of replication, allowing for iterative chromosomal manipulations of the same strain, as is often required in metabolic engineering. In addition, we demonstrate the ability to efficiently cure the second plasmid in the system by targeting with Cas9, leaving the cells plasmid-free.Shell Global Solutions (US)National Institute of Food and Agriculture (U.S.) (Postdoctoral Fellowship 2013-67012-21022

Scarless Cas9 Assisted Recombineering (no‐SCAR) in Escherichia coli, an Easy‐to‐Use System for Genome Editing

The discovery and development of genome editing systems that leverage the site‐specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a “guide” RNA to enable the Cas9 nuclease to make a double‐strand break at a particular genome locus, which is repaired by non‐homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology‐directed repair. However, E. coli lacks robust systems for double‐strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9‐mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ‐Red system for recombination in E. coli, we created a highly efficient system for marker‐free and scarless genome editing.National Institute of Food and Agriculture (U.S.) (Award 2013-67012-21022)United States. Army Research Office (Grant W911NF-09-0001

'GR 7' Grape

'GR 7' is an early / mid-season red wine grape for use primarily in red wine blends. It is distinguished from other red wine grapes grown in cool climates by its high degree of winter hardiness, adaptation to mechanized production systems, and ability to survive in older plantings where other red wine grapes are lost due to tomato and tobacco ringspot virus infections. ?GR 7? is a highly productive, easy to manage cultivar, and is the sixth wine grape to be developed by the New York State Agricultural Experiment Station of Cornell University

Use of differential thermal analysis to quantify bud cold hardiness of grape selections and clones

Differential thermal analysis (DTA) was used to characterize primary bud mid-winter cold hardiness of Vitis spp. Bud hardiness reached a maximum and was rather stable during the months of January and February at Geneva, New York. Because cold tolerance increases during periods of prolonged cold, observed freezing temperature was adjusted on the basis of the freezing temperature of cv. Concord on the day of observation. DTA gives reproducible and meaningful estimates of bud freezing temperature. Such data account for at least 50 % of the among-cultivar variance in overall vine cold hardiness

Remaily Seedless Grape

Since the late 19th century when grape breeding began at the New York State Agricultural Experiment Station, a major goal has been to combine certain fruit attributes such as seedlessness, crisp texture, and adherent skin of Vitis vinifera L. table grapes with some of the vegetative characters such as disease resistance and cold hardiness of native American hybrid (V. labruscana, Bailey) grape cultivars

Herding Livestock – the Phoenix Rises from the Ashes? Digital Herding as a Future Tool for Grazing Livestock

Today, sustainable management of grazing livestock requires high efforts in management and fencing. Nowadays, several developments in digital technologies for herding grazing animals are arising. We conducted a systematic review on current developments in digital technologies for managing grazing animals within the landscape. We mainly focused on cattle (Bos taurus) and sheep (Ovis aries). We highlight the most promising developments of virtual fencing used in recent research to evaluate effectiveness, animal behaviour and welfare. Moreover, we highlight current research in digital herding by drones and robots. We discuss the potential and current limitations of digital tools for sustainable grazing management. Recent study results showed that virtual fences are highly efficient in keeping cattle within allocated pasture areas. So far, there has been no evidence for harmful impacts on animal welfare or reduction in animal performance. First findings suggest that drones can also herd and move animals. However, knowledge on the efficiency and potential effects on animal welfare when using drones is limited. First findings have shown that robots are able to gather animals to a specific location and heart-rate and blood tests showed that the animals were less stressed by the robot than they were by a human. However, research on herding drones and robots is still in its infancy. Digital tools provide the opportunity for precise livestock movement control and could facilitate the implementation of both productive and biodiversity-friendly grazing

Analysis of the relationship between grapevine cultivars, sports and clones via DNA fingerprinting

DNA fingerprinting utilizing RAPD polymorphisms was employed to investigate the relationship among 16 grapevine cultivars and sports thought to have arisen from these cultivars. From 53 primers, a total of 464 bands were generated, of which 29 % were common to all genotypes tested. Cluster analysis classified all tested cultivars into two main groups (Vitis vinifera L. and V. x Labruscana Bailey) as expected. No polymorphism was detected among known clones of Chardonnay (Ch. clone 7, Ch. clone 78 and Ch. Geneva clone) or Pinot noir (P. n. clone 29, P. n. Geneva clone and P. n. Pernand). Pinot Meunier, Pinot gris, and Gamay Beaujolais displayed patterns indistinguishable from Pinot noir. Auxerrois and Melon showed unique patterns and may be classified as distinct cultivars. Chardonnay clone 7 shared 84 % of its bands with Pinot noir. There was more than 97 % RAPD amplicon homology between Niagara and two supposed sports, and between Concord and a red-fruited sport. Taking into account the error rate in scoring RAPD bands, the evidence is against the hypothesis that the three sports are distinct cultivars. While RAPD banding patterns could not distinguish between known clones, they were useful for distinguishing between phenotypically similar cultivars and for assessing the origins of cultivars thought to have originated as sports

Minimally invasive keyhole approaches in spinal intradural tumor surgery: report of two cases and conceptual considerations

Despite their histologically benign nature, intradural tumors may become symptomatic by virtue of their space-occupying effect, causing severe neurological deficits. The gold standard treatment is total excision of the lesion; however, extended dorsal and dorsolateral approaches may cause late complications due to iatrogenic destruction of the posterolateral elements of the spine. In this article, Axel Perneczky’s concept of minimally invasive spinal tumor surgery is described. Two illustrative cases demonstrate the feasibility and safety of keyhole fenestrations exposing the spinal canal. The first case is a 67-year-old woman with a 1-year history of severe back pain, right thoracic radiculopathy, and slight gait disturbance. Neuroimaging revealed a right-sided combined intradural-extradural spinal schwannoma at T11/12 , with compression of the spinal cord and lateral extension through the intervertebral foramen. The tumor was successfully removed through a contralateral left-sided hemilaminectomy. The second case is a 38-year-old man with progressive spinal ataxia caused by an intramedullary C6-8 ependymoma. Here, bi-segmental interlaminar fenestrations were performed, allowing safe and minimally invasive tumor resection. In both cases, postoperative imaging confirmed complete tumor removal and both patients showed neither neurological deterioration, nor vertebral instability or pain syndromes

Functional consequences of seven novel mutations in the CYP11B1 Gene: four mutations associated with nonclassic and three mutations causing classic 11 -Hydroxylase Deficiency

Context: Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency. Objective: The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations. Methods: We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein. Results: All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11β-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively. Conclusion: Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling i