BATCH-GE : batch analysis of next-generation sequencing data for genome editing assessment

Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data. Therefore, we designed BATCH-GE, an easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data, available from https://github.com/WouterSteyaert/BATCH-GE.git. BATCH-GE detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel. Furthermore, this new tool provides flexibility by allowing the user to adapt a number of input variables. The performance of BATCH-GE was evaluated in two genome editing experiments, aiming to generate knock-out and knock-in zebrafish mutants. This tool will not only contribute to the evaluation of CRISPR/Cas9-based experiments, but will be of use in any genome editing experiment and has the ability to analyze data from every organism with a sequenced genome

Transparent and Shaped Stiffness Reflection for Telesurgery

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CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments

Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined

Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients

Ascorbate requiring Fe2+/2-oxoglutarate-dependent dioxygenases located in the nucleoplasm have been shown to participate in epigenetic regulation of gene expression via histone and DNA demethylation. Transport of dehydroascorbic acid is impaired in the endomembranes of fibroblasts from arterial tortuosity syndrome (ATS) patients, due to the mutation in the gene coding for glucose transporter GLUT10. We hypothesized that altered nuclear ascorbate concentration might be present in ATS fibroblasts, affecting dioxygenase activity and DNA demethylation. Therefore, our aim was to characterize the subcellular distribution of vitamin C, the global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine levels, and the effect of ascorbate supplementation in control and ATS fibroblast cultures. Diminished nuclear accumulation of ascorbate was found in ATS fibroblasts upon ascorbate or dehydroascorbic acid addition. Analyzing DNA samples of cultured fibroblasts from controls and ATS patients, a lower global 5-hydroxymethylcytosine level was found in ATS fibroblasts, which could not be significantly modified by ascorbate addition. Investigation of the (hydroxy)methylation status of specific regions in six candidate genes related to ascorbate metabolism and function showed that ascorbate addition could stimulate hydroxymethylation and active DNA demethylation at the PPAR-gamma gene region in control fibroblasts only. The altered DNA hydroxymethylation patterns in patient cells both at the global level and at specific gene regions accompanied with decreased nuclear accumulation of ascorbate suggests the epigenetic role of vitamin C in the pathomechanism of ATS. The present findings represent the first example for the role of vitamin C transport in epigenetic regulation suggesting that ATS is a compartmentalization disease

Glucose transporter type 10—lacking in arterial tortuosity syndrome—facilitates dehydroascorbic acid transport

Loss-of-function mutations in the gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS), a rare connective tissue disorder. In this study GLUT10-mediated dehydroascorbic acid (DAA) transport was investigated, supposing its involvement in the pathomechanism. GLUT10 protein produced by in vitro translation and incorporated into liposomes efficiently transported DAA. Silencing of GLUT10 decreased DAA transport in immortalized human fibroblasts whose plasma membrane was selectively permeabilized. Similarly, the transport of DAA through endomembranes was markedly reduced in fibroblasts from ATS patients. Re-expression of GLUT10 in patients’ fibroblasts restored DAA transport activity. The present results demonstrate that GLUT10 is a DAA transporter and DAA transport is diminished in the endomembranes of fibroblasts from ATS patients

GLUT10-Lacking in Arterial Tortuosity Syndrome-Is Localized to the Endoplasmic Reticulum of Human Fibroblasts.

GLUT10 belongs to a family of transporters that catalyze the uptake of sugars/polyols by facilitated diffusion. Loss-of-function mutations in the SLC2A10 gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS). Since subcellular distribution of the transporter is dubious, we aimed to clarify the localization of GLUT10. In silico GLUT10 localization prediction suggested its presence in the endoplasmic reticulum (ER). Immunoblotting showed the presence of GLUT10 protein in the microsomal, but not in mitochondrial fractions of human fibroblasts and liver tissue. An even cytosolic distribution with an intense perinuclear decoration of GLUT10 was demonstrated by immunofluorescence in human fibroblasts, whilst mitochondrial markers revealed a fully different decoration pattern. GLUT10 decoration was fully absent in fibroblasts from three ATS patients. Expression of exogenous, tagged GLUT10 in fibroblasts from an ATS patient revealed a strict co-localization with the ER marker protein disulfide isomerase (PDI). The results demonstrate that GLUT10 is present in the ER

A Mechatronic and Model-Mediated Approach to Bilateral Teleoperation LoTESS: Towards a Force Feedback Telesurgical System (Een mechatronische en model-gebaseerde aanpak van bilaterale teleoperatie LoTESS: een telechirurgisch systeem met krachtterugkoppeling)

People have always been fascinated by the ability to interact with an environment or with another person at a distance or behind a barrier. In minimally invasive surgery, the body wall is an example of such a barrier, since the surgeon loses his/her direct vision and manipulation ability. To reduce the additional complexity of minimally invasive surgery, teleoperation systems have entered the surgical theatre. These systems provide the surgeon with a feeling of immersion into the surgical workspace by restoring the natural hand-eye coordination. Nowadays, these systems are frequently used, despite a total lack of haptic feedback, often mentioned as their major drawback. Bilateral teleoperation, which restores the sense of touch, constitutes the core of this PhD thesis. It allows a person to manipulate an environment via a robotic slave device while force feedback is provided through a master device. The challenge of bilateral teleoperation is to find a good balance between the often conflicting requirements for performance and stability. The first part of this thesis presents a systematic analysis of the coupled stability properties of two state-of-the-art force sensing-based controllers. The analysis uses the classical two-port passivity and absolute stability methods along with in this thesis proposed bounded environment passivity method. The latter allows the specification of a model structure for the environment as well as the inclusion of bounds on the environment parameters. The analysis quantifies the effect of rigid-body dynamics, structural flexibilities, control parameters and signal filters, along with their interconnectivity. Based on this analysis, rules of thumb are defined for the master and slave hardware design as well as for the control design. The second part of the thesis describes the development of LoTESS, a teleoperation system for keyhole surgery that provides reliable force feedback. The force feedback requirement was the primary concern during the development. Design decisions are made according to the aforementioned rules of thumb for hardware and control design. LoTESS consists of three subsystems that are also valuable as separate units: a new 3-d.o.f. robotic device that can be used as a haptic joystick, a robotic device for keyhole surgery that uses a passive trocar support device to obtain a controlled motion of the instrument tip and an extracorporeal force measurement system. The combination of these subsystems with a force-sensing based controller and an adaptive rule for the damping at the master, results in a system that is both transparent in free space and stable in contact with the envisioned environments. The last part of this thesis looks into scenarios where the rules of thumb for hardware and control design cannot be applied, i.e. scenarios that are characterised by the presence of an inevitable and significant time or phase lag between the master and slave. Model-mediated teleoperation is presented as a powerful alternative for these challenging scenarios. The models are interpreted as time-varying signals and from this perspective, the stability of model-mediated teleoperation is discussed. Several case studies are described that consider models of various complexity, including an object location, a linear stiffness and a plane with variable location and orientation. These case studies are used to illustrate the concepts of the predictive power of a model, model switching, enhanced transparency and sensor fusion for model-estimation. Preview of LoTESS: http://www.youtube.com/watch?v=zWjs3Sr3tuInrpages: 340status: publishe

Stability of Model-Mediated Teleoperation: Discussion and Experiments

The design of a bilateral teleoperation system remains chal- lenging in cases with high-impedance slave robots or substantial commu- nication delays. Especially for these scenarios, model-mediated teleoper- ation offers a promising new approach. In this paper, we present a first stability discussion. We examine the continuous behavior using general control principles and discuss how the model structure and its predictive power affects system lag and stability. We also recognize the unavoidabil- ity of discrete model jumps and discuss measures to isolate events and prevent limit cycles. The discussions are illustrated in a single degree of freedom case and supported by single degree of freedom experiments.status: publishe