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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 65,403 bioRxiv papers from 289,703 authors.

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in category bioengineering

1,283 results found. For more information, click each entry to expand.

21: Summary of muscle parameters for Hill-based muscle modeling in the human lower limb
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Posted to bioRxiv 02 Dec 2016

Summary of muscle parameters for Hill-based muscle modeling in the human lower limb
2,501 downloads bioengineering

Ross H Miller

A summary is presented of five mechanical parameters from human lower limb skeletal muscles critical for Hill-based muscle modeling: the optimal fiber length, the fiber pennation angle, the physiological cross-sectional area (PCSA), the unloaded tendon length, and the fast-twitch fiber fraction. The data presented are drawn from a total of 29 publications including human cadaver studies, in vivo imaging studies of live humans, musculoskeletal modeling studies, and combinations of these methods. Where possible, parameter values were adjusted from the referenced data to present them with consistent definitions (normalization of measured fiber lengths to optimal sarcomere length, and calculation of PCSA as the ratio of fiber volume to fiber length). It is seen that within a specific muscle, optimal fiber lengths are fairly consistent between studies, pennation angles and PCSAs vary widely between studies, and data for unloaded tendon length are comparatively sparse. Few studies have reported fiber type fractions for a large number of muscles. Guidelines for implementing these parameter values in muscle modeling and musculoskeletal modeling are suggested.

22: No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice
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Posted to bioRxiv 09 Feb 2018

No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice
2,489 downloads bioengineering

Vivek Iyer, Katharina Boroviak, Mark Thomas, Brendan Doe, Edward Ryder, David Adams

CRISPR-Cas technologies have transformed genome-editing of experimental organisms and have immense therapeutic potential. Despite significant advances in our understanding of the CRISPR-Cas9 system, concerns remain over the potential for off-target effects. Recent studies have addressed these concerns using whole-genome sequencing (WGS) of gene-edited embryos or animals to search for de novo mutations (DNMs), which may represent candidate changes induced by poor editing fidelity. Critically, these studies used strain-matched but not pedigree-matched controls and thus were unable to reliably distinguish generational or colony-related differences from true DNMs. Here we used a trio design and whole genome sequenced 8 parents and 19 embryos, where 10 of the embryos were mutagenised with well-characterised gRNAs targeting the coat colour Tyrosinase (Tyr) locus. Detailed analyses of these whole genome data allowed us to conclude that if CRISPR mutagenesis were causing SNV or indel off-target mutations in treated embryos, then the number of these mutations is not statistically distinguishable from the background rate of DNMs occurring due to other processes.

23: Silk assembly integrates cells into a 3D fibrillar network that promotes cell spreading and proliferation
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Posted to bioRxiv 29 Aug 2018

Silk assembly integrates cells into a 3D fibrillar network that promotes cell spreading and proliferation
2,385 downloads bioengineering

Ulrika Johansson, Mona Widhe, Nancy Dekki Shalaly, Irene Linares Arregui, Linnea Nilebäck, Christos Panagiotis Tasiopoulos, Carolina Åstrand, Per-Olof Berggren, Christian Gasser, My Hedhammar

Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) fibrillar network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into fibrillar networks under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed fibrils. In the resulting 3D scaffold, the cells proliferate and spread out with tissue-like morphology. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk fibers with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables both differentiation of stem- or precursor cells in 3D and facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a fibrillary ECM-like network, with potential as base for engineering of functional tissue.

24: PERFORMANCE OF AN AMBULATORY DRY-EEG DEVICE FOR AUDITORY CLOSED-LOOP STIMULATIONS IN THE HOME ENVIRONMENT
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Posted to bioRxiv 25 Sep 2017

PERFORMANCE OF AN AMBULATORY DRY-EEG DEVICE FOR AUDITORY CLOSED-LOOP STIMULATIONS IN THE HOME ENVIRONMENT
2,378 downloads bioengineering

E. Debellemaniere, S. Chambon, C. Pinaud, V. Thorey, D. Léger, M. Chennaoui, P.J. Arnal, M.N. Galtier

Objective. Recent research has shown that auditory closed-loop stimulations can enhance sleep slow oscillations (SO) to improve N3 sleep quality and cognition. Previous studies have been conducted in a lab environment and on a small sample size. The present study aimed at validating and assessing the performance of a novel ambulatory wireless dry-EEG device (WDD), for auditory closed-loop stimulations of SO during N3 sleep at home. Material and Methods. The performance of the WDD to detect N3 sleep automatically and to send auditory closed-loop stimulations on SO were tested on 20 young healthy subjects who slept with both the WDD and a miniaturized polysomnography (part 1) in both stimulated and sham nights within a double blind, randomized and crossover design. The electrophysiological effects of auditory closed-loop stimulation on delta power increase were assessed after 1 and 10 nights of stimulations on an observational pilot study in the home environment including 90 middle-aged subjects (part 2). Results. The sensitivity and specificity of the WDD to automatically detect N3 sleep in real-time were 0.70 and 0.90, respectively. The stimulation accuracy of the SO ascending-phase targeting was 45+/-52 degrees. The stimulation protocol induced an increase of 39.5 % of delta power after the stimulations. The increase of SO response to auditory stimulations remained at the same level after 10 consecutive nights. . Conclusion. The WDD shows good performances to automatically detect in real-time N3 sleep and to send auditory closed-loop stimulations on SO accurately. These stimulations increased the SO amplitude during N3 sleep without any adaptation effect after 10 consecutive nights. This tool provides new perspectives to figure out novel sleep EEG biomarkers in longitudinal studies and can be interesting to conduct broad studies on the effects of auditory stimulations during sleep.

25: Genetically encoded ratiometric indicators for potassium ion
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Posted to bioRxiv 25 Jan 2018

Genetically encoded ratiometric indicators for potassium ion
2,371 downloads bioengineering

Yi Shen, Sheng-Yi Wu, Vladimir Rancic, Yong Qian, Shin-Ichiro Miyashita, Klaus Ballanyi, Robert E Campbell, Min Dong

Potassium ion (K+) homeostasis and dynamics play critical roles in regulating various biological activities, and the ability to monitor K+ spatial-temporal dynamics is critical to understanding these biological functions. Here we report the design and characterization of a Förster resonance energy transfer (FRET)-based genetically encoded K+ indicator, KIRIN1, constructed by inserting a bacterial cytosolic K+ binding protein (Kbp) between a fluorescent protein (FP) FRET pair, mCerulean3 and cp173Venus. Binding of K+ induces a conformational change in Kbp, resulting in an increase in FRET efficiency. KIRIN1 was able to detect K+ at physiologically relevant concentrations in vitro and is highly selective toward K+ over Na+. We further demonstrated that KIRIN1 allowed real-time imaging of pharmacologically induced depletion of cytosolic K+ in live cells, and KIRIN1 also enabled optical tracing of K+ efflux and reuptake in neurons upon glutamate stimulation in cultured primary neurons. These results demonstrate that KIRIN1 is a valuable tool to detect K+ in vitro and in live cells.

26: High Numerical Aperture Epi-illumination Selective Plane Illumination Microscopy
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Posted to bioRxiv 28 Feb 2018

High Numerical Aperture Epi-illumination Selective Plane Illumination Microscopy
2,346 downloads bioengineering

Bin Yang, Yina Wang, Siyu Feng, Veronica Pessino, Nico Stuurman, Bo Huang

Selective-plane illumination microscopy (SPIM) provides unparalleled advantages for long-term volumetric imaging of living organisms. In order to achieve high-resolution imaging in common biological sample holders, we designed a high numerical aperture (NA) epi-illumination SPIM (eSPIM) system, which utilizes a single objective and has an identical sample interface as an inverted fluorescence microscope with no additional reflection elements. This system has an effective detection NA of > 1.06. We demonstrated multicolor and fast volumetric imaging of live cells and single-molecule super-resolution microscopy using our system.

27: On the relationship between indenation hardness and modulus, and the damage resistance of biological materials.
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Posted to bioRxiv 09 Feb 2017

On the relationship between indenation hardness and modulus, and the damage resistance of biological materials.
2,345 downloads bioengineering

David Labonte, Anne-Kristin Lenz, Michelle L Oyen

The remarkable mechanical performance of biological materials is based on intricate structure-function relationships. Nanoindentation has become the primary tool for characterising biological materials, as it allows to relate structural changes to variations in mechanical properties on small scales. However, the respective theoretical background and associated interpretation of the parameters measured via indentation derives largely from research on "traditional" engineering materials such as metals or ceramics. Here, we discuss the functional relevance of indentation hardness in biological materials by presenting a meta-analysis of its relationship with indentation modulus. Across seven orders of magnitude, indentation hardness was directly proportional to indentation modulus, illustrating that hardness is not an independent material property. Using a lumped parameter model to deconvolute indentation hardness into components arising from reversible and irreversible deformation, we establish criteria which allow to interpret differences in indentation hardness across or within biological materials. The ratio between hardness and modulus arises as a key parameter, which is a proxy for the ratio between irreversible and reversible deformation during indentation, and the material's yield strength. Indentation hardness generally increases upon material dehydration, however to a larger extend than expected from accompanying changes in indentation modulus, indicating that water acts as a "plasticiser". A detailed discussion of the role of indentation hardness, modulus and toughness in damage control during sharp or blunt indentation yields comprehensive guidelines for a performance-based ranking of biological materials, and suggests that quasi-plastic deformation is a frequent yet poorly understood damage mode, highlighting an important area of future research.

28: Machine learning-guided channelrhodopsin engineering enables minimally-invasive optogenetics
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Posted to bioRxiv 03 Mar 2019

Machine learning-guided channelrhodopsin engineering enables minimally-invasive optogenetics
2,327 downloads bioengineering

Claire N Bedbrook, Kevin K Yang, J. Elliott Robinson, Viviana Gradinaru, Frances H Arnold

We have engineered light-gated channelrhodopsins (ChRs) whose current strength and light sensitivity enable minimally-invasive neuronal circuit interrogation. Current ChR tools applied to the mammalian brain require intracranial surgery for transgene delivery and implantation of invasive fiber-optic cables to produce light-dependent activation of a small volume of brain tissue [~1 mm3]. To enable optogenetics for large brain volumes and without the need for invasive implants, our ChR engineering approach leverages the significant literature of ChR variants to train statistical models for the design of new, high-performance ChRs. With Gaussian Process models trained on a limited experimental set of 102 functionally characterized ChR variants, we designed high-photocurrent ChRs with unprecedented light sensitivity; three of these, ChRger1, ChRger2, and ChRger3, enable optogenetic activation of the nervous system via minimally-invasive systemic transgene delivery with rAAV-PHP.eB, which was not possible previously due to low per-cell transgene copy produced by systemic delivery. These engineered ChRs enable light-induced neuronal excitation without invasive intracranial surgery for virus delivery or fiber optic implantation, i.e. they enable minimally-invasive optogenetics.

29: Directed evolution of split APEX peroxidase
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Posted to bioRxiv 25 Oct 2018

Directed evolution of split APEX peroxidase
2,319 downloads bioengineering

Yisu Han, Jeffrey D. Martell, Tess C Branon, Daniela Boassa, David M Shechner, Mark H. Ellisman, Alice Y. Ting

APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications, from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). Twenty rounds of FACS-based selections from yeast-displayed fragment libraries, using three different yeast display configurations, produced a 200-amino acid N-terminal fragment (with 9 mutations relative to APEX2) called AP and a 50-amino acid C-terminal fragment called EX. AP and EX fragments were each inactive on their own but reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within telomerase noncoding RNA, and at mitochondria-endoplasmic reticulum contact sites.

30: Ultrasound Imaging of Gene Expression in Mammalian Cells
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Posted to bioRxiv 18 Mar 2019

Ultrasound Imaging of Gene Expression in Mammalian Cells
2,311 downloads bioengineering

Arash Farhadi, Gabrielle H. Ho, Daniel P. Sawyer, Raymond W. Bourdeau, MIkhail G Shapiro

The study of cellular processes occurring inside intact organisms and the development of cell-based diagnostic and therapeutic agents requires methods to visualize cellular functions such as gene expression in deep tissues. Ultrasound is a widely used biomedical technology enabling deep-tissue imaging with high spatial and temporal resolution. However, no genetically encoded molecular reporters are available to connect ultrasound contrast to gene expression in mammalian cells. To address this limitation, we introduce the first mammalian acoustic reporter genes. Starting with an eleven-gene polycistronic gene cluster derived from bacteria, we engineered a eukaryotic genetic program whose introduction into mammalian cells results in the expression of a unique class of intracellular air-filled protein nanostructures called gas vesicles. The scattering of ultrasound by these nanostructures allows mammalian cells to be visualized at volumetric densities below 0.5%, enables the monitoring of dynamic circuit-driven gene expression, and permits high-resolution imaging of gene expression in living animals. These mammalian acoustic reporter genes enable previously impossible approaches to monitoring the location, viability and function of mammalian cells in vivo.

31: Open Design 3D-Printable Adjustable Micropipette that meets ISO Standard for Accuracy
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Posted to bioRxiv 20 Feb 2017

Open Design 3D-Printable Adjustable Micropipette that meets ISO Standard for Accuracy
2,295 downloads bioengineering

Martin Brennan, Fahad F. Bokhari, David Eddington

Scientific communities are drawn to the open source model as an increasingly utilitarian method to produce and share work. Initially used as a means to develop freely available soft- ware, open source projects have been applied to hardware including scientific tools. Increasing convenience of 3D printing has fueled the proliferation of open labware projects aiming to develop and share designs for scientific tools that can be produced in-house as cheap alter- natives to commercial products. We present our design of a micropipette that is assembled from 3D-printable parts and some hardware that works by actuating a disposable syringe to a user adjustable limit. Graduations on the syringe are used to accurately adjust the set point to the desired volume. Our open design printed micropipette is assessed in comparison to a commercial pipette and meets ISO 8655 standards.

32: Improved Split Fluorescent Proteins For Endogenous Protein Labeling
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Posted to bioRxiv 12 May 2017

Improved Split Fluorescent Proteins For Endogenous Protein Labeling
2,284 downloads bioengineering

Siyu Feng, Sayaka Sekine, Veronica Pessino, Han Li, Manuel D. Leonetti, Bo Huang

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split-GFP1-10/11; as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split-sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.

33: Evaluating different passive optical clearing protocols for two-photon deep tissue imaging in adult intact visceral and neuronal organs
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Posted to bioRxiv 27 Apr 2015

Evaluating different passive optical clearing protocols for two-photon deep tissue imaging in adult intact visceral and neuronal organs
2,260 downloads bioengineering

R.C Vlieg, C Gillespie, W M Steve Lee

Imaging cellular activities in an entire intact whole organ with light is a grand challenge in optical microscopy. To date, most passive clearing techniques were shown to transform brain, neuronal and embryo tissue into near transparent state for deep tissue imaging. Here, we expand these passive clearing protocol from neuronal tissue (brain and spinal cord) to other visceral organs such as liver and colon and further evaluate their ?depth-clearing performance? based on image contrast of endogenous fluorescence structures. We found that the SeeDB achieve highest depth in brain, 3DISCO is adept at clearing liver and spinal cord and ScaleViewA2 in colon. Overall, 3DISCO clears more rapidly than other agents at a higher cost, while ScaleViewA2 is the most economical but clears at a slower rate. This study, for the first time, provide a direct evaluation of imaging depth, cost and time amongst passive tissue clearing protocols for different intact organs.

34: A 3D-printed hand-powered centrifuge for molecular biology
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Posted to bioRxiv 16 Jan 2019

A 3D-printed hand-powered centrifuge for molecular biology
2,150 downloads bioengineering

Gaurav Byagathvalli, Aaron Pomerantz, Soham Sinha, Janet Standeven, M. Saad Bhamla

The centrifuge is an essential tool for many aspects of research and medical diagnostics. However, conventional centrifuges are often inaccessible outside of conventional laboratory settings, such as remote field sites, require a constant external power source, and can be prohibitively costly in resource-limited settings and STEM-focused programs. Here we present the 3D-Fuge, a 3D-printed hand-powered centrifuge, as a novel alternative to standard benchtop centrifuges. Based on the design principles of a paper-based centrifuge, this 3D-printed instrument increases the volume capacity to 2 mL and can reach hand-powered centrifugation speeds up to 6,000 rpm. The 3D-Fuge devices presented here are capable of centrifugation of a wide variety of different solutions such as spinning down samples for biomarker applications and performing nucleotide extractions as part of a portable molecular lab setup. We introduce the design and proof-of-principle trials that demonstrate the utility of low-cost 3D printed centrifuges for use in remote and educational settings.

35: A three-dimensional culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction and disease modeling
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Posted to bioRxiv 02 Mar 2018

A three-dimensional culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction and disease modeling
2,085 downloads bioengineering

Mohsen Afshar Bakooshli, Ethan S Lippmann, Ben Mulcahy, Nisha R Iyer, Christine T Nguyen, Kayee Tung, Bryan A Stewart, Hubrecht van den Dorpel, Tobias Fuehrmann, Molly S. Shoichet, Anne Bigot, Elena Pegoraro, Henry Ahn, Howard Ginsberg, Mei Zhen, Randolph S Ashton, Penney M. Gilbert

Two-dimensional (2D) human skeletal muscle fiber cultures are ill equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections within two weeks. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium transient imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-culture. This demonstrates that the 3D co-culture system supports a developmental shift from the embryonic to adult form of the receptor that does not occur in 2D co-culture. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. This work delivers a simple, reproducible, and adaptable method to model and evaluate adult human NMJ de novo development and disease in culture.

36: Peptide sequencing in an electrolytic cell with two nanopores in tandem and exopeptidase
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Posted to bioRxiv 17 Feb 2015

Peptide sequencing in an electrolytic cell with two nanopores in tandem and exopeptidase
2,048 downloads bioengineering

G Sampath

A nanopore-based approach to peptide sequencing without labels or immobilization is considered. It is based on a tandem cell (RSC Adv., 2015, 5, 167-171) with the structure [cis1, upstream pore (UNP), trans1/cis2, downstream pore (DNP), trans2]. An amino or carboxyl exopeptidase attached to the downstream side of UNP cleaves successive leading residues in a peptide threading from cis1 through UNP. A cleaved residue translocates to and through DNP where it is identified. A Fokker-Planck model is used to compute translocation statistics for each amino acid type. Multiple discriminators, including a variant of the current blockade level and translocation times through trans1/cis2 and DNP, identify a residue. Calculations show the 20 amino acids to be grouped by charge (+, -, neutral) and ordered within each group (which makes error correction easier). The minimum cleaving interval required of the exopeptidase, the sample size (number of copies of the peptide to sequence or runs with one copy) to identify a residue with a given confidence level, and confidence levels for a given sample size are calculated. The results suggest that if the exopeptidase cleaves each and every residue and does so in a reasonable time, peptide sequencing with acceptable (and correctable) errors may be feasible. If validated experimentally the proposed device could be an alternative to mass spectrometry and gel electrophoresis. Implementation-related issues are discussed.

37: Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole
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Posted to bioRxiv 26 Jan 2019

Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole
2,043 downloads bioengineering

Aisha Yesbolatova, Toyoaki Natsume, Ken-ichiro Hayashi, Masato T Kanemaki

Controlling protein expression using a degron is advantageous because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15-45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR-Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.

38: Recent Advancement in Biosensors Technology for Animal and Livestock Health Management
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Posted to bioRxiv 19 Apr 2017

Recent Advancement in Biosensors Technology for Animal and Livestock Health Management
2,041 downloads bioengineering

Suresh Neethirajan, Sheng-Tung Huang, Satish K. Tuteja, David Kelton

The term biosensors encompasses devices that have the potential to quantify physiological, immunological and behavioural responses of livestock and multiple animal species. Novel biosensing methodologies offer highly specialized monitoring devices for the specific measurement of individual and multiple parameters covering an animal physiology as well as monitoring of an animals environment. These devices are not only highly specific and sensitive for the parameters being analysed, but they are also reliable and easy to use, and can accelerate the monitoring process. Novel biosensors in livestock management provide significant benefits and applications in disease detection and isolation, health monitoring and detection of reproductive cycles, as well as monitoring physiological well-being of the animal via analysis of the animals environment. With the development of integrated systems and the Internet of Things, the continuously monitoring devices are expected to become affordable. The data generated from integrated livestock monitoring is anticipated to assist farmers and the agricultural industry to improve animal productivity in the future. The data is expected to reduce the impact of the livestock industry on the environment, while at the same time driving the new wave towards the improvements of viable farming techniques. This review focuses on the emerging technological advancements in monitoring of livestock health for detailed, precise information on productivity, as well as physiology and well-being. Biosensors will contribute to the 4th revolution in agriculture by incorporating innovative technologies into cost-effective diagnostic methods that can mitigate the potentially catastrophic effects of infectious outbreaks in farmed animals.

39: A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types
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Posted to bioRxiv 28 Mar 2019

A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types
2,024 downloads bioengineering

David N. Nguyen, Theodore L. Roth, Jonathan Li, Peixin Amy Chen, Murad R. Mamedov, Linda T Vo, Victoria Tobin, Ryan Apathy, Daniel Goodman, Eric Shifrut, Jeffrey A Bluestone, Jennifer M Puck, Francis C Szoka, Alexander Marson

Virus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies. We recently developed a non-viral CRISPR-Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells. Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to 'shuttle' the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for non-viral genome reprogramming that we successfully applied in regulatory T cells (Tregs), γδ-T cells, B cells, NK cells, and primary and iPS-derived hematopoietic stem progenitor cells (HSPCs).

40: High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis
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Posted to bioRxiv 19 Mar 2018

High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis
1,974 downloads bioengineering

Derek M Mason, Cedric R Weber, Cristina Parola, Simon M Meng, Victor Greiff, William J Kelton, Sai T Reddy

Antibody engineering is performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offers advantages associated with expression in their final therapeutic format (full-length glycosylated IgG), however, the inability to express large and diverse libraries severely limits their potential throughput. To address this limitation, we have developed homology-directed mutagenesis (HDM), a novel method which extends the concept of CRISPR/Cas9-mediated homology-directed repair (HDR). HDM leverages oligonucleotides with degenerate codons to generate site-directed mutagenesis libraries in mammalian cells. By improving HDM efficiency (>35-fold) and combining mammalian display screening with next-generation sequencing (NGS), we validated this approach can be used for key applications in antibody engineering at high-throughput: rational library construction, novel variant discovery, affinity maturation, and deep mutational scanning (DMS). We anticipate that HDM will be a valuable tool for engineering and optimizing antibodies in mammalian cells, and eventually enable directed evolution of other complex proteins and cellular therapeutics.

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