Development of CRISPR/Cas9-based Gene Drive Biotechnology in S. Cerevisiae PDF Download
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Author: Yao Yan
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
The budding yeast Saccharomyces cerevisiae is a well-studied eukaryotic model organism for investigating the fundamental aspects of molecular and cellular biology. It also can be used to evaluate the technical feasibility of new technologies in eukaryotic cells. In our lab, we aimed to use Saccharomyces cerevisiae as a model to develop CRISPR/Cas9-based gene drive biotechnology. The CRISPR/Cas9 biotechnology is a powerful gene editing tool used to modify target genomic sequences in all forms of living creatures including animals, plants, fungi, and bacteria. One potential application of this molecular method is within a "gene drive" system. This unique arrangement of CRISPR within a genome may one day allow for global control of biological populations and be used to eliminate pests, parasites, and invasive species. However, there are many concerns regarding the utilization of this technology, including gene drive design, control, and development of resistance, etc. In our study, we developed an artificial gene drive system in budding yeast, which could edit multiple loci at the same time. We demonstrated that this triple gene drive system could successfully edit three DNA targets independently with only a single copy of S. pyogenes Cas9. We also found that the occurrence of NHEJ could be repressed by modifying DNA Ligase IV. However, successful gene drives still allowed for the occurrence of a small number of "resistant" clones. We investigated potential causes of this imperfect drive activity. Our work illustrated that imperfect activation of the inducible promoter driving expression of the Cas9 nuclease or issues with multiplexing to artificial sequences may have resulted in a small percentage of resistant/inactive clones. The CRISPR/Cas9 system can also be used to regulate gene transcription. This involves a mutated Cas9 variant that has lost its nuclease activity (dCas9). We developed a CRISPR/dCas9 system by tagging dCas9 with transcriptional regulators. Our experiments demonstrated that CRISPR/dCas9 could activate target gene transcription when tagged with the transcriptional activator VPR and repress gene transcription when tagged with the transcriptional repressor Mxi1.
Author: Yao Yan
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
The budding yeast Saccharomyces cerevisiae is a well-studied eukaryotic model organism for investigating the fundamental aspects of molecular and cellular biology. It also can be used to evaluate the technical feasibility of new technologies in eukaryotic cells. In our lab, we aimed to use Saccharomyces cerevisiae as a model to develop CRISPR/Cas9-based gene drive biotechnology. The CRISPR/Cas9 biotechnology is a powerful gene editing tool used to modify target genomic sequences in all forms of living creatures including animals, plants, fungi, and bacteria. One potential application of this molecular method is within a "gene drive" system. This unique arrangement of CRISPR within a genome may one day allow for global control of biological populations and be used to eliminate pests, parasites, and invasive species. However, there are many concerns regarding the utilization of this technology, including gene drive design, control, and development of resistance, etc. In our study, we developed an artificial gene drive system in budding yeast, which could edit multiple loci at the same time. We demonstrated that this triple gene drive system could successfully edit three DNA targets independently with only a single copy of S. pyogenes Cas9. We also found that the occurrence of NHEJ could be repressed by modifying DNA Ligase IV. However, successful gene drives still allowed for the occurrence of a small number of "resistant" clones. We investigated potential causes of this imperfect drive activity. Our work illustrated that imperfect activation of the inducible promoter driving expression of the Cas9 nuclease or issues with multiplexing to artificial sequences may have resulted in a small percentage of resistant/inactive clones. The CRISPR/Cas9 system can also be used to regulate gene transcription. This involves a mutated Cas9 variant that has lost its nuclease activity (dCas9). We developed a CRISPR/dCas9 system by tagging dCas9 with transcriptional regulators. Our experiments demonstrated that CRISPR/dCas9 could activate target gene transcription when tagged with the transcriptional activator VPR and repress gene transcription when tagged with the transcriptional repressor Mxi1.
Author: Sean Edward Gomez Guy
Publisher:
ISBN:
Category :
Languages : en
Pages : 48
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Book Description
Vector-borne diseases pose a significant health threat to millions. Genetic modification through CRISPR/Cas9-based gene drive technologies may be able to solve this problem by rapidly spreading disease resistance throughout vector populations. However, the long-term effects of active gene drive elements at evolutionary scales has yet to be extensively explored. To address this issue, we constructed S. cerevisiae strains that express both Cas9 and gRNA, Cas9 and no gRNA, or neither Cas9 nor gRNA. We founded replicate populations and propagated these over 1000 generations. From whole-population, whole-genome sequences, we identified changes in allele frequency in each population. By comparing the numbers of alleles to appear or fix in each population, we find evidence of higher mutation rates in strains expressing Cas9 nuclease. In parallel, we directly measure the mutation rate differences due to gene drive or Cas9 alone. Our work underscores the need to control CRISPR/Cas9 activity to limit unintended effects in natural populations.
Author: Jiazhang Lian
Publisher: Frontiers Media SA
ISBN: 2889664058
Category : Science
Languages : en
Pages : 254
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Book Description
Author: Luis María Vaschetto
Publisher: CRC Press
ISBN: 1000540812
Category : Mathematics
Languages : en
Pages : 268
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Book Description
The CRISPR-Cas9 genome-editing system is creating a revolution in the science world. In the laboratory, CRISPR-Cas9 can efficiently be used to target specific genes, correct mutations and regulate gene expression of a wide array of cells and organisms, including human cells. CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases is a unique reading material for college students, academicians, and other health professionals interested in learning about the broad range of applications of CRISPR/Cas9 genetic scissors. Some topics included in this book are: the role of the CRISPR/Cas9 system in neuroscience, gene therapy, epigenome editing, genome mapping, cancer, virus infection control strategies, regulatory challenges and bioethical considerations.
Author: National Academies of Sciences, Engineering, and Medicine
Publisher: National Academies Press
ISBN: 0309437873
Category : Science
Languages : en
Pages : 231
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Book Description
Research on gene drive systems is rapidly advancing. Many proposed applications of gene drive research aim to solve environmental and public health challenges, including the reduction of poverty and the burden of vector-borne diseases, such as malaria and dengue, which disproportionately impact low and middle income countries. However, due to their intrinsic qualities of rapid spread and irreversibility, gene drive systems raise many questions with respect to their safety relative to public and environmental health. Because gene drive systems are designed to alter the environments we share in ways that will be hard to anticipate and impossible to completely roll back, questions about the ethics surrounding use of this research are complex and will require very careful exploration. Gene Drives on the Horizon outlines the state of knowledge relative to the science, ethics, public engagement, and risk assessment as they pertain to research directions of gene drive systems and governance of the research process. This report offers principles for responsible practices of gene drive research and related applications for use by investigators, their institutions, the research funders, and regulators.
Author: Congressional Service
Publisher:
ISBN: 9781793024428
Category :
Languages : en
Pages : 41
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Book Description
Scientists have long sought the ability to control and modify DNA--the code of life. A gene editing technology known as CRISPR-Cas9 offers the potential for substantial improvement over other gene editing technologies in that it is simple to use and inexpensive and has a relatively high degree of precision and efficiency. These characteristics have led many in the scientific and business communities to assert that CRISPR-Cas9 will lead to groundbreaking advances in many fields, including agriculture, energy, ecosystem conservation, and the investigation, prevention, and treatment of diseases. Over the next 5 to 10 years, the National Academy of Sciences projects a rapid increase in the scale, scope, complexity, and development rate of biotechnology products, many enabled by CRISPR-Cas9. Concomitant with the promise of potential benefits, such advances may pose new risks and raise ethical concerns. For example, a Chinese researcher recently claimed that he had created the first genetically engineered human babies. According to the researcher, he used CRISPR-Cas9 to disable a gene that will make it harder for the twin girls, who were born in November 2018, to contract human immunodeficiency virus (HIV). The as yet unsubstantiated claim has sparked outrage and ethical debates by the international scientific community and others. Prior use of CRISPR-Cas9 gene editing in human embryos was generally limited to nonviable embryos, in part, to address ethical concerns such as the fact that the genetic change would affect not only the immediate patient, but also future generations who would inherit the change. Additionally, CRISPR-related approaches (gene drives) are being considered to reduce or eliminate the mosquito that serves as the primary vector for the transmission of Zika or malaria, thereby improving public health. Some scientists and environmental groups have raised ethical questions and expressed concerns about the unintended ecological consequences of eliminating a species or introducing a genetically modified organism into an open environment. Some experts assert that the current system for regulating biotechnology products--the Coordinated Framework for the Regulation of Biotechnology--may be inadequate, with the potential to leave gaps in oversight. Regulatory gaps may lead to increased uncertainty that could affect the development of future biotechnology products or a loss of public confidence in the ability of regulators to ensure that such products are safe. In the 116th Congress, policymakers may want to examine the potential benefits and risks associated with the use of CRISPR-Cas9 gene editing, including the ethical, social, and legal implications of CRISPR-related biotechnology products. Congress also may have a role to play with respect to regulation, research and development, and economic competitiveness associated with CRISPR-Cas9 gene editing and future biotechnology products.
Author: Valeria Mapelli
Publisher: Humana Press
ISBN: 9781493905621
Category : Science
Languages : en
Pages : 0
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Book Description
Yeast Metabolic Engineering: Methods and Protocols provides the widely established basic tools used in yeast metabolic engineering, while describing in deeper detail novel and innovative methods that have valuable potential to improve metabolic engineering strategies in industrial biotechnology applications. Beginning with an extensive section on molecular tools and technology for yeast engineering, this detailed volume is not limited to methods for Saccharomyces cerevisiae, but describes tools and protocols for engineering other yeasts of biotechnological interest, such as Pichia pastoris, Hansenula polymorpha and Zygosaccharomyces bailii. Tools and technologies for the investigation and determination of yeast metabolic features are described in detail as well as metabolic models and their application for yeast metabolic engineering, while a chapter describing patenting and regulations with a special glance at yeast biotechnology closes the volume. Written in the highly successful Methods in Molecular Biology series format, most chapters include an introduction to their respective topic, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols and tips on troubleshooting and avoiding known pitfalls. Comprehensive and authoritative, Yeast Metabolic Engineering: Methods and Protocols aims to familiarize researchers with the current state of these vital and increasingly useful technologies.
Author: Vijai Singh
Publisher: Academic Press
ISBN: 0128181419
Category : Science
Languages : en
Pages : 386
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Book Description
Genome Engineering via CRISPR-Cas9 Systems presents a compilation of chapters from eminent scientists from across the globe who have established expertise in working with CRISPR-Cas9 systems. Currently, targeted genome engineering is a key technology for basic science, biomedical and industrial applications due to the relative simplicity to which they can be designed, used and applied. However, it is not easy to find relevant information gathered in a single source. The book contains a wide range of applications of CRISPR in research of bacteria, virus, algae, plant and mammalian and also discusses the modeling of drosophila, zebra fish and protozoan, among others. Other topics covered include diagnosis, sensor and therapeutic applications, as well as ethical and regulatory issues. This book is a valuable source not only for beginners in genome engineering, but also researchers, clinicians, stakeholders, policy makers, and practitioners interested in the potential of CRISPR-Cas9 in several fields. Provides basic understanding and a clear picture on how to design, use and implement the CRISPR-Cas9 system in different organisms Explains how to create an animal model for disease research and screening purposes using CRISPR Discusses the application of CRISPR-Cas9 systems in basic sciences, biomedicine, virology, bacteriology, molecular biology, neurology, cancer, industry, and many more
Author:
Publisher: Academic Press
ISBN: 0323853226
Category : Science
Languages : en
Pages : 244
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Book Description
Reprogramming the Genome: Applications of CRISPR-Cas in Non-mammalian Systems, Part A presents a collation of chapters written by global, eminent scientists. CRISPR-Cas9 system is an RNA-mediated immune system of bacteria and archaea that protects from bacteriophage infections. It is one of the revolutionized technologies to uplift biology to the next stages. Chapters in this release include An Introduction and applications of CRISPR-Cas Systems, History, evolution and classification of CRISPR-Cas associated systems, CRISPR based bacterial genome editing and removal of pathogens, CRISPR based genome editing and removal of human viruses, CRISPR based development of RNA editing and diagnostic platform, and much more. Additional sections cover Genome engineering in insects for control of vector borne diseases, Development of insect cell line using CRISPR technology, CRISPRing protozoan parasites to better understand the biology of diseases, CRISPR based genome editing of Caenorhabditis elegans, and a variety of other important topics. Offers a basic understanding and clear picture of genome editing CRISPR-Cas systems in different organisms Explains how to create an animal model for disease diagnosis/research and reprogram CRISPR for removal of virus, bacteria, fungi, protozoan, and many more Discusses the advances, patents, applications, challenges and opportunities in CRISPR-Cas9 systems in basic sciences, biomedicine, virology, bacteriology, molecular biology, and many more
Author: Miguel Fernández-Niño
Publisher: BoD – Books on Demand
ISBN: 1839696389
Category : Science
Languages : en
Pages : 106
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Book Description
The current advances in sequencing, data mining, DNA synthesis, cloning, in silico modeling, and genome editing have opened a new field of research known as Synthetic Genomics. The main goal of this emerging area is to engineer entire synthetic genomes from scratch using pre-designed building blocks obtained by chemical synthesis and rational design. This has opened the possibility to further improve our understanding of genome fundamentals by considering the effect of the whole biological system on biological function. Moreover, the construction of non-natural biological systems has allowed us to explore novel biological functions so far not discovered in nature. This book summarizes the current state of Synthetic Genomics, providing relevant examples in this emerging field.
