scholarly journals Genome-driven cell engineering review: in vivo and in silico metabolic and genome engineering

2019 ◽  
Vol 63 (2) ◽  
pp. 267-284 ◽  
Author(s):  
Sophie Landon ◽  
Joshua Rees-Garbutt ◽  
Lucia Marucci ◽  
Claire Grierson
Keyword(s):  
Computational Models ◽  
Genome Engineering ◽  
Computational Design ◽  
Biological Research ◽  
Cellular Processes ◽  
Recent Developments ◽  
Entire Cell ◽  
Design Algorithms ◽  
Genome Design

Abstract Producing ‘designer cells’ with specific functions is potentially feasible in the near future. Recent developments, including whole-cell models, genome design algorithms and gene editing tools, have advanced the possibility of combining biological research and mathematical modelling to further understand and better design cellular processes. In this review, we will explore computational and experimental approaches used for metabolic and genome design. We will highlight the relevance of modelling in this process, and challenges associated with the generation of quantitative predictions about cell behaviour as a whole: although many cellular processes are well understood at the subsystem level, it has proved a hugely complex task to integrate separate components together to model and study an entire cell. We explore these developments, highlighting where computational design algorithms compensate for missing cellular information and underlining where computational models can complement and reduce lab experimentation. We will examine issues and illuminate the next steps for genome engineering.

scholarly journals A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling

2020 ◽  
Author(s):  
Deepti Trivedi ◽  
Vinitha CM ◽  
Karishma Bisht ◽  
Vishnu Janardan ◽  
Awadhesh Pandit ◽  
...  
Keyword(s):  
Human Disease ◽  
Genome Engineering ◽  
Cultured Cells ◽  
Cellular Organization ◽  
Cellular Processes ◽  
Lipid Signalling ◽  
Disease Biology ◽  
A Genome ◽  
Biochemical Analyses

SummaryPhosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signalling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signalling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here we describe a set of CRISPR based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

scholarly journals A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Eline D. Postma ◽  
Sofia Dashko ◽  
Lars van Breemen ◽  
Shannara K. Taylor Parkins ◽  
Marcel van den Broek ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Engineering ◽  
De Novo ◽  
Transcriptional Unit ◽  
Dna Assembly ◽  
Cellular Processes ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Expression Platform

ABSTRACTThe construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes Synthetic Chromosomes (SynChs) as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular SynChs of 50 and 100 Kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. SynChs made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding SynChs were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.

scholarly journals Designing Minimal Genomes Using Whole-Cell Models

2018 ◽  
Author(s):  
Joshua Rees ◽  
Oliver Chalkley ◽  
Sophie Landon ◽  
Oliver Purcell ◽  
Lucia Marucci ◽  
...  
Keyword(s):  
In Silico ◽  
Genome Engineering ◽  
Cell Model ◽  
Mycoplasma Genitalium ◽  
Computational Design ◽  
Functional Assay ◽  
Proof Of Concept ◽  
Computational Tools ◽  
Whole Cell ◽  
Genome Design

AbstractIn the future, entire genomes tailored to specific functions and environments could be designed using computational tools. However, computational tools for genome design are currently scarce. Here we present algorithms that enable the use of design-simulate-test cycles for genome design, using genome minimisation as a proof-of-concept. Minimal genomes are ideal for this purpose as they have a simple functional assay, the cell either replicates or not. We used the first (and currently only published) whole-cell model, for the bacterium Mycoplasma genitalium. Our computational design-simulate-test cycles discovered novel in-silico minimal genomes smaller than JCVI-Syn3.0, a bacteria with, currently, the smallest genome that can be grown in pure culture. In the process, we identified 10 low essentiality genes, 18 high essentiality genes, and produced evidence for at least two Mycoplasma genitalium in-silico minimal genomes. This work brings combined computational and laboratory genome engineering a step closer.

scholarly journals A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Deepti Trivedi ◽  
Vinitha CM ◽  
Karishma Bisht ◽  
Vishnu Janardan ◽  
Awadhesh Pandit ◽  
...  
Keyword(s):  
Human Disease ◽  
Genome Engineering ◽  
Cultured Cells ◽  
Cellular Organization ◽  
Cellular Processes ◽  
Disease Biology ◽  
A Genome ◽  
Biochemical Analyses

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here, we describe a set of CRISPR-based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

scholarly journals CRISPR/Cas: Advances, Limitations, and Applications for Precision Cancer Research

2021 ◽  
Vol 8 ◽  
Author(s):  
Yue Yang ◽  
Jin Xu ◽  
Shuyu Ge ◽  
Liqin Lai
Keyword(s):  
Cancer Research ◽  
Targeted Delivery ◽  
Genome Engineering ◽  
Resistance Mechanisms ◽  
Rna Sequences ◽  
Dna And Rna ◽  
Recent Developments ◽  
Associated Proteins ◽  
Cancer Tumor

Cancer is one of the most leading causes of mortalities worldwide. It is caused by the accumulation of genetic and epigenetic alterations in 2 types of genes: tumor suppressor genes (TSGs) and proto-oncogenes. In recent years, development of the clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized genome engineering for different cancer research ranging for research ranging from fundamental science to translational medicine and precise cancer treatment. The CRISPR/CRISPR associated proteins (CRISPR/Cas) are prokaryote-derived genome editing systems that have enabled researchers to detect, image, manipulate and annotate specific DNA and RNA sequences in various types of living cells. The CRISPR/Cas systems have significant contributions to discovery of proto-oncogenes and TSGs, tumor cell epigenome normalization, targeted delivery, identification of drug resistance mechanisms, development of high-throughput genetic screening, tumor models establishment, and cancer immunotherapy and gene therapy in clinics. Robust technical improvements in CRISPR/Cas systems have shown a considerable degree of efficacy, specificity, and flexibility to target the specific locus in the genome for the desired applications. Recent developments in CRISPRs technology offers a significant hope of medical cure against cancer and other deadly diseases. Despite significant improvements in this field, several technical challenges need to be addressed, such as off-target activity, insufficient indel or low homology-directed repair (HDR) efficiency, in vivo delivery of the Cas system components, and immune responses. This study aims to overview the recent technological advancements, preclinical and perspectives on clinical applications of CRISPR along with their advantages and limitations. Moreover, the potential applications of CRISPR/Cas in precise cancer tumor research, genetic, and other precise cancer treatments discussed.

CRISPR Gene Editing on Human Embryos

2020 ◽  
Author(s):  
Fatemeh Sefid ◽  
Saedeh Khadempar ◽  
Roshanak Shamriz ◽  
Nooshin Amjadi
Keyword(s):  
Genome Editing ◽  
Human Embryo ◽  
Genome Engineering ◽  
Human Reproduction ◽  
Human Embryos ◽  
Gene Therapies ◽  
Recent Developments ◽  
Technical Possibility ◽  
Early Human

Background: With the recent development of CRISPR/Cas9 genome editing technology, the possibility to genetically influence the human germline (gametes and embryos) has become a separate technical possibility. As a powerful skill for genome engineering, the CRISPR/Cas9 system has been effectively applied to adjust the genomes of several species. The purpose of this review was to appraise the technology and build concepts for the launch of precise hereditary modifications in early human embryos.   Methods: We conducted a systematic review of the related literatures searched from PubMed, Google scholar, Web of Science up to June 30, 2017 and then we extracted the essential data. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and significant challenges and advances in the field as a comprehensive practical guide to absorbed users of genome editing technologies. We introduce factors that influence CRISPR/Cas9 efficacy which must be addressed before effective in vivo human embryo therapy can be realized .in this review, we highlight the advancements that have been made using CRISPR/Cas9 in relation to Human Embryo.   Results and Conclusion: The possibility of CRISPR/Cas9 use in the context of human reproduction, to change embryos, germline cells, and pluripotent stem cells are studied created on the writers' expert belief. We discuss recent developments leading to the operation of Human Embryonic gene therapies in clinical trials and consider the predictions for future advances in this rapidly developing field.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

scholarly journals Persistent luminescence nanoparticles for cancer theranostics application

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Nian Liu ◽  
Xiao Chen ◽  
Xia Sun ◽  
Xiaolian Sun ◽  
Junpeng Shi
Keyword(s):  
Uv Light ◽  
Combined Therapy ◽  
Tumor Therapy ◽  
High Sensitivity ◽  
Persistent Luminescence ◽  
Therapeutic Drugs ◽  
High Penetration ◽  
Recent Developments ◽  
Cancer Theranostics

AbstractPersistent luminescence nanoparticles (PLNPs) are unique optical materials that emit afterglow luminescence after ceasing excitation. They exhibit unexpected advantages for in vivo optical imaging of tumors, such as autofluorescence-free, high sensitivity, high penetration depth, and multiple excitation sources (UV light, LED, NIR laser, X-ray, and radiopharmaceuticals). Besides, by incorporating other functional molecules, such as photosensitizers, photothermal agents, or therapeutic drugs, PLNPs are also widely used in persistent luminescence (PersL) imaging-guided tumor therapy. In this review, we first summarize the recent developments in the synthesis and surface functionalization of PLNPs, as well as their toxicity studies. We then discuss the in vivo PersL imaging and multimodal imaging from different excitation sources. Furthermore, we highlight PLNPs-based cancer theranostics applications, such as fluorescence-guided surgery, photothermal therapy, photodynamic therapy, drug/gene delivery and combined therapy. Finally, future prospects and challenges of PLNPs in the research of translational medicine are also discussed.

scholarly journals A Novel Method to Predict Drug-Target Interactions Based on Large-Scale Graph Representation Learning

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2111
Author(s):  
Bo-Wei Zhao ◽  
Zhu-Hong You ◽  
Lun Hu ◽  
Zhen-Hao Guo ◽  
Lei Wang ◽  
...  
Keyword(s):  
Drug Target ◽  
Large Scale ◽  
Computational Models ◽  
Structural Information ◽  
Characteristic Curve ◽  
Representation Learning ◽  
Graph Representation ◽  
Convolutional Network ◽  
Novel Method

Identification of drug-target interactions (DTIs) is a significant step in the drug discovery or repositioning process. Compared with the time-consuming and labor-intensive in vivo experimental methods, the computational models can provide high-quality DTI candidates in an instant. In this study, we propose a novel method called LGDTI to predict DTIs based on large-scale graph representation learning. LGDTI can capture the local and global structural information of the graph. Specifically, the first-order neighbor information of nodes can be aggregated by the graph convolutional network (GCN); on the other hand, the high-order neighbor information of nodes can be learned by the graph embedding method called DeepWalk. Finally, the two kinds of feature are fed into the random forest classifier to train and predict potential DTIs. The results show that our method obtained area under the receiver operating characteristic curve (AUROC) of 0.9455 and area under the precision-recall curve (AUPR) of 0.9491 under 5-fold cross-validation. Moreover, we compare the presented method with some existing state-of-the-art methods. These results imply that LGDTI can efficiently and robustly capture undiscovered DTIs. Moreover, the proposed model is expected to bring new inspiration and provide novel perspectives to relevant researchers.

miR-125b enhances metastasis and progression of cancer via the TXNIP and HIF1α pathway in pancreatic cancer

2021 ◽  
pp. 1-12
Author(s):  
Pengli Wang ◽  
Dan Zheng ◽  
Hongyang Qi ◽  
Qi Gao
Keyword(s):  
Pancreatic Cancer ◽  
Hypoxia Inducible Factor ◽  
Immunofluorescence Assay ◽  
Luciferase Assay ◽  
Interacting Protein ◽  
Over Expression ◽  
Thioredoxin Interacting Protein ◽  
Cellular Processes ◽  
And Migration

BACKGROUND: MicroRNAs (miRNAs) play potential role in the development of various types of cancer conditions including pancreatic cancer (PC) targeting several cellular processes. Present study was aimed to evaluate function of miR-125b and the mechanism involved in PC. METHODS: Cell migration, MTT and BrdU study was done to establish the migration capability, cell viability and cell proliferation respectively. Binding sites for miR-125b were recognized by luciferase assay, expression of protein by western blot and immunofluorescence assay. In vivo study was done by BALB/c nude xenograft mice for evaluating the function of miR-125b. RESULTS: The study showed that expression of miR-125b was elevated in PC cells and tissues, and was correlated to proliferation and migration of cells. Also, over-expression of miR-125b encouraged migration, metastasis and proliferation of BxPC-3 cells, the suppression reversed it. We also noticed that thioredoxin-interacting protein (TXNIP) was the potential target of miR-125b. The outcomes also suggested that miR-125b governed the expression of TXNIP inversely via directly attaching to the 3′-UTR activating hypoxia-inducible factor 1α (HIF1α). Looking into the relation between HIF1α and TXNIP, we discovered that TXNIP caused the degradation and export of HIF1α by making a complex with it. CONCLUSION: The miR-125b-TXNIP-HIF1α pathway may serve useful strategy for diagnosing and treating PC.

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