Matching Items (13)

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CRISPR Based Synthetic Transcription Factors: The Future of Transcriptional Therapeutics

Description

Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the

Pinpoint control over endogenous gene expression in vivo has long been a fevered dream for clinicians and researchers alike. With the recent repurposing of programmable, RNA-guided DNA endonucleases from the CRISPR bacterial immune system, this dream is becoming a powerful reality. Engineered CRISPR based transcriptional regulators have enabled researchers to perturb endogenous gene expression in vivo, allowing for the therapeutic reprogramming of cell and tissue behavior. However, for this technology to be of maximal use, a variety of technological hurdles still need to be addressed. Here, we discuss recent advances and integrative strategies that can help pave the way towards a new class of transcriptional therapeutics.

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Date Created
  • 2019-05

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Safe CRISPR: Challenges and Opportunities

Description

Conservatism is intrinsic to safety of emerging biotechnologies. Fear of unintended consequences, misuse, and bioterror are rightfully essential in our discussions of novel innovations. Clustered regularly Interspaced Short Palindromic Repeats

Conservatism is intrinsic to safety of emerging biotechnologies. Fear of unintended consequences, misuse, and bioterror are rightfully essential in our discussions of novel innovations. Clustered regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated proteins are no exception. This review will characterize environmental and health-related risks of CRISPR-applications and expound upon mechanisms that are or can be used to minimize risk. CRISPR is broadening access and simplifying genomic and transcriptomic editing leading to wide-range usage in all of biology. Utilization in gene therapies, gene drives, and agriculture could all be universally impactful applications that need their own safety technologies and guidelines. The initial ethical guidelines and recommendations, that will guide these technologies, are being steadily developed. However, technical advances are required to facilitate safe usage. Since the advent of CRISPR gene editing in 2012 advances to limit off-target edits (both cellular and genomic) have been developed. Delivery systems that use viral or nanoparticle packaging incorporate safety mechanisms to guard against undesirable side effects are being produced and rigorously tested. Besides its applications in basic biology and potential as a gene therapy, CRISPR had humbler beginnings. Industrially it was, albeit unknowingly, used to fend off infection in productions of yogurt batches. This was one of the earliest applications of CRISPR, however with the knowledge we now have ecological and industrial uses of CRISPR have multiplied. Gene drives have the power to spread genetic mutations throughout populations and agricultural uses to better crop genomes are also of interest. These uses have struck a chord with interest groups (environmentalists, anti-GMO groups, etc) who imagine how this technology can drastically alter species with unforeseen evolutionary changes that could reshape present-day ecosystems. This review will describe existing technologies that will safeguard humanity and its interests while fully employing CRISPRs far-reaching potentiality.

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Date Created
  • 2018-05

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Analyzing Uncertainties Around Gene Drives: A Case Study of Mosquitos in Sub-Saharan Africa

Description

Malaria is a disease that has plagued human populations throughout history. Malaria is cause by the parasite Plasmodium, which uses mosquitoes as a vector for transfer. Current methods for controlling

Malaria is a disease that has plagued human populations throughout history. Malaria is cause by the parasite Plasmodium, which uses mosquitoes as a vector for transfer. Current methods for controlling malaria include issuing bed nets to citizens, spraying home with insecticides, and reactive medical care. However, using Clustered Regularly Interspaced Short Palindromic repeats (CRISPR) in conjunction with the Cas9 protein found in bacteria, the genomes of mosquitoes can be edited to remove the ability of mosquitoes to host Plasmodium or to create sex bias in which the birth rate of males is increased so as to make reproduction near impossible. Using CRISPR, this genome edit can be ‘driven’ through a population by increasing the likelihood of that gene being passed onto subsequent generations until the entire population possesses that gene; a gene drive can theoretically be used to eliminate malaria around the world. This paper identifies uncertainties concerning scientific, environmental, governance, economic ,and social aspects of researching and implementing gene drives and makes recommendations concerning these areas for the emerging technology of gene drives concerning the eradication of malaria using Sub-Saharan Africa as a case study

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Date Created
  • 2017-05

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RNA-Guided Modification of Synthetic Gene Networks Using CRISPR-Cas Systems

Description

The ability to edit chromosomal regions is an important tool for the study of gene function and the ability to engineer synthetic gene networks. CRISPR-Cas systems, a bacterial RNA-guided immune

The ability to edit chromosomal regions is an important tool for the study of gene function and the ability to engineer synthetic gene networks. CRISPR-Cas systems, a bacterial RNA-guided immune system against foreign nucleic acids, have recently been engineered for a plethora of genome engineering and transcriptional regulation applications. Here we employ engineered variants of CRISPR systems in proof-of-principle experiments demonstrating the ability of CRISPR-Cas derived single-DNA-strand cutting enzymes (nickases) to direct host-cell genomic recombination. E.coli is generally regarded as a poorly recombinogenic host with double-stranded DNA breaks being highly lethal. However, CRISPR-guided nickase systems can be easily programmed to make very precise, non-lethal, incisions in genomic regions directing both single reporter gene and larger-scale recombination events deleting up to 36 genes. Genome integrated repetitive elements of variable sizes can be employed as sites for CRISPR induced recombination. We project that single-stranded based editing methodologies can be employed alongside preexisting genome engineering techniques to assist and expedite metabolic engineering and minimalized genome research.

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Date Created
  • 2014-05

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Plasmid Design for Making a HEK293t Reporter Cell Line to Study Gene Expression Dynamics

Description

Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or

Cell fate is a complex and dynamic process with many genetic components. It has often been likened to “multistable” mathematical systems because of the numerous possible “stable” states, or cell types, that cells may end up in. Due to its complexity, understanding the process of cell fate and differentiation has proven challenging. A better understanding of cell differentiation has applications in regenerative stem cell therapies, disease pathologies, and gene regulatory networks.
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.

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Agent

Created

Date Created
  • 2019-05

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Increased Enrichment and Generation of Isogenic Lines Using a Transient Reporter for Editing Enrichment

Description

Alzheimer’s disease (AD) affects over 5 million individuals each year in the United States. Furthermore, most cases of AD are sporadic, making it extremely difficult to model and study in

Alzheimer’s disease (AD) affects over 5 million individuals each year in the United States. Furthermore, most cases of AD are sporadic, making it extremely difficult to model and study in vitro. CRISPR/Cas9 and base editing technologies have been of recent interest because of their ability to create single nucleotide edits at nearly any genomic sequence using a Cas9 protein and a guide RNA (sgRNA). Currently, there is no available phenotype to differentiate edited cells from unedited cells. Past research has employed fluorescent proteins bound to Cas9 proteins to attempt to enrich for edited cells, however, these methods are only reporters of transfection (RoT) and are no indicative of actual base-editing occurring. Thus, this study proposes a transient reporter for editing enrichment (TREE) and Cas9-mediated adenosine TREE (CasMasTREE) which use plasmids to co-transfect with CRISPR/Cas9 technologies to serve as an indicator of base-editing. Specifically, TREE features a blue fluorescent protein (BFP) mutant that, upon a C-T conversion, changes the emission spectrum to a green fluorescent protein (GFP). CasMasTREE features a mCherry and GFP protein separated by a stop codon which can be negated using an A-G conversion. By employing a sgRNA that targets one of the TREE plasmids and at least one genomic site, cells can be sorted for GFP(+) cells. Using these methods, base-edited isogenic hiPSC line generation using TREE (BIG-TREE) was created to generate isogenic hiPSC lines with AD-relevant edits. For example, BIG-TREE demonstrates the capability of converting Apolipoprotein E (APOE), a gene associated with AD-risk development, wildtype (3/3) into another isoform, APOE2/2, to create isogenic hiPSC lines. The capabilities of TREE are vast and can be applied to generate various models of diseases with specific genomic edits.

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Agent

Created

Date Created
  • 2020

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Development of CRISPR-RNA guided recombinases for genome engineering

Description

Recombinases are powerful tools for genome engineering and synthetic biology, however recombinases are limited by a lack of user-programmability and often require complex directed-evolution experiments to retarget specificity. Conversely, CRISPR

Recombinases are powerful tools for genome engineering and synthetic biology, however recombinases are limited by a lack of user-programmability and often require complex directed-evolution experiments to retarget specificity. Conversely, CRISPR systems have extreme versatility yet can induce off-target mutations and karyotypic destabilization. To address these constraints we developed an RNA-guided recombinase protein by fusing a hyperactive mutant resolvase from transposon TN3 to catalytically inactive Cas9. We validated recombinase-Cas9 (rCas9) function in model eukaryote Saccharomyces cerevisiae using a chromosomally integrated fluorescent reporter. Moreover, we demonstrated cooperative targeting by CRISPR RNAs at spacings of 22 or 40bps is necessary for directing recombination. Using PCR and Sanger sequencing, we confirmed rCas9 targets DNA recombination. With further development we envision rCas9 becoming useful in the development of RNA-programmed genetic circuitry as well as high-specificity genome engineering.

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Agent

Created

Date Created
  • 2018

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Engineering Open Chromatin with Synthetic Pioneer Factors:: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin

Description

Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an

Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin.

I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions.

SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug.

Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.

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Agent

Created

Date Created
  • 2019

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Developing a CRISPR-Mediated Knockout TCR Human T Cell Line for Use in Cloning Antigen-Specific T Cell Receptors

Description

Adoptive transfer of T cells engineered to express synthetic antigen-specific T cell receptors (TCRs) has provocative therapeutic applications for treating cancer. However, expressing these synthetic TCRs in a CD4+ T

Adoptive transfer of T cells engineered to express synthetic antigen-specific T cell receptors (TCRs) has provocative therapeutic applications for treating cancer. However, expressing these synthetic TCRs in a CD4+ T cell line is a challenge. The CD4+ Jurkat T cell line expresses endogenous TCRs that compete for space, accessory proteins, and proliferative signaling, and there is the potential for mixed dimer formation between the α and β chains of the endogenous receptor and that of the synthetic cancer-specific TCRs. To prevent hybridization between the receptors and to ensure the binding affinity measured with flow cytometry analysis is between the tetramer and the TCR construct, a CRISPR-Cas9 gene editing pipeline was developed. The guide RNAs (gRNAs) within the complex were designed to target the constant region of the α and β chains, as they are conserved between TCR clonotypes. To minimize further interference and confer cytotoxic capabilities, gRNAs were designed to target the CD4 coreceptor, and the CD8 coreceptor was delivered in a mammalian expression vector. Further, Golden Gate cloning methods were validated in integrating the gRNAs into a CRISPR-compatible mammalian expression vector. These constructs were transfected via electroporation into CD4+ Jurkat T cells to create a CD8+ knockout TCR Jurkat cell line for broadly applicable uses in T cell immunotherapies.

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Agent

Created

Date Created
  • 2020

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Generation of isogenic pluripotent stem cell lines for study of APOE, an Alzheimer’s risk factor

Description

Alzheimer’s disease (AD), despite over a century of research, does not have a clearly defined pathogenesis for the sporadic form that makes up the majority of disease incidence. A variety

Alzheimer’s disease (AD), despite over a century of research, does not have a clearly defined pathogenesis for the sporadic form that makes up the majority of disease incidence. A variety of correlative risk factors have been identified, including the three isoforms of apolipoprotein E (ApoE), a cholesterol transport protein in the central nervous system. ApoE ε3 is the wild-type variant with no effect on risk. ApoE ε2, the protective and most rare variant, reduces risk of developing AD by 40%. ApoE ε4, the risk variant, increases risk by 3.2-fold and 14.9-fold for heterozygous and homozygous representation respectively. Study of these isoforms has been historically complex, but the advent of human induced pluripotent stem cells (hiPSC) provides the means for highly controlled, longitudinal in vitro study. The effect of ApoE variants can be further elucidated using this platform by generating isogenic hiPSC lines through precise genetic modification, the objective of this research. As the difference between alleles is determined by two cytosine-thymine polymorphisms, a specialized CRISPR/Cas9 system for direct base conversion was able to be successfully employed. The base conversion method for transitioning from the ε3 to ε2 allele was first verified using the HEK293 cell line as a model with delivery via electroporation. Following this verification, the transfection method was optimized using two hiPSC lines derived from ε4/ε4 patients, with a lipofection technique ultimately resulting in successful base conversion at the same site verified in the HEK293 model. Additional research performed included characterization of the pre-modification genotype with respect to likely off-target sites and methods of isolating clonal variants.

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Agent

Created

Date Created
  • 2017