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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 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.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis advisor) / Brafman, David A (Committee member) / Tian, Xiao-jun (Committee member) / Arizona State University (Publisher)
Created2018
Description
A novel clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool for simultaneous gene editing and regulation was designed and tested. This study used the CRISPR-associated protein 9 (Cas9) endonuclease in complex with a 14-nucleotide (nt) guide RNA (gRNA) to repress a gene of interest using the Krüppel associated box (KRAB) domain, while also performing a separate gene modification using a 20-nt gRNA targeted to a reporter vector. DNA Ligase IV (LIGIV) was chosen as the target for gene repression, given its role in nonhomologous end joining, a common DNA repair process that competes with the more precise homology-directed repair (HDR).
To test for gene editing, a 20-nt gRNA was designed to target a disrupted enhanced green fluorescent protein (EGFP) gene present in a reporter vector. After the gRNA introduced a double-stranded break, cells attempted to repair the cut site via HDR using a DNA template within the reporter vector. In the event of successful gene editing, the EGFP sequence was restored to a functional state and green fluorescence was detectable by flow cytometry. To achieve gene repression, a 14-nt gRNA was designed to target LIGIV. The gRNA included a com protein recruitment domain, which recruited a Com-KRAB fusion protein to facilitate gene repression via chromatin modification of LIGIV. Quantitative polymerase chain reaction was used to quantify repression.
This study expanded upon earlier advancements, offering a novel and versatile approach to genetic modification and transcriptional regulation using CRISPR/Cas9. The overall results show that both gene editing and repression were occurring, thereby providing support for a novel CRISPR/Cas system capable of simultaneous gene modification and regulation. Such a system may enhance the genome engineering capabilities of researchers, benefit disease research, and improve the precision with which gene editing is performed.
To test for gene editing, a 20-nt gRNA was designed to target a disrupted enhanced green fluorescent protein (EGFP) gene present in a reporter vector. After the gRNA introduced a double-stranded break, cells attempted to repair the cut site via HDR using a DNA template within the reporter vector. In the event of successful gene editing, the EGFP sequence was restored to a functional state and green fluorescence was detectable by flow cytometry. To achieve gene repression, a 14-nt gRNA was designed to target LIGIV. The gRNA included a com protein recruitment domain, which recruited a Com-KRAB fusion protein to facilitate gene repression via chromatin modification of LIGIV. Quantitative polymerase chain reaction was used to quantify repression.
This study expanded upon earlier advancements, offering a novel and versatile approach to genetic modification and transcriptional regulation using CRISPR/Cas9. The overall results show that both gene editing and repression were occurring, thereby providing support for a novel CRISPR/Cas system capable of simultaneous gene modification and regulation. Such a system may enhance the genome engineering capabilities of researchers, benefit disease research, and improve the precision with which gene editing is performed.
ContributorsChapman, Jennifer E (Author) / Kiani, Samira (Thesis advisor) / Ugarova, Tatiana (Thesis advisor) / Marchant, Gary (Committee member) / Arizona State University (Publisher)
Created2018
Description
The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli.
1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin.
2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists.
ContributorsDaer, René (Author) / Haynes, Karmella (Thesis advisor) / Brafman, David (Committee member) / Nielsen, David (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2017
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 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.
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.
ContributorsBarrett, Cassandra M (Author) / Haynes, Karmella A (Thesis advisor) / Rege, Kaushal (Committee member) / Mills, Jeremy (Committee member) / Kiani, Samira (Committee member) / Arizona State University (Publisher)
Created2019
Description
Currently in synthetic biology only the Las, Lux, and Rhl quorum sensing pathways have been adapted for broad engineering use. Quorum sensing allows a means of cell to cell communication in which a designated sender cell produces quorum sensing molecules that modify gene expression of a designated receiver cell. While useful, these three quorum sensing pathways exhibit a nontrivial level of crosstalk, hindering robust engineering and leading to unexpected effects in a given design. To address the lack of orthogonality among these three quorum sensing pathways, previous scientists have attempted to perform directed evolution on components of the quorum sensing pathway. While a powerful tool, directed evolution is limited by the subspace that is defined by the protein. For this reason, we take an evolutionary biology approach to identify new orthogonal quorum sensing networks and test these networks for cross-talk with currently-used networks. By charting characteristics of acyl homoserine lactone (AHL) molecules used across quorum sensing pathways in nature, we have identified favorable candidate pathways likely to display orthogonality. These include Aub, Bja, Bra, Cer, Esa, Las, Lux, Rhl, Rpa, and Sin, which we have begun constructing and testing. Our synthetic circuits express GFP in response to a quorum sensing molecule, allowing quantitative measurement of orthogonality between pairs. By determining orthogonal quorum sensing pairs, we hope to identify and adapt novel quorum sensing pathways for robust use in higher-order genetic circuits.
ContributorsMuller, Ryan (Author) / Haynes, Karmella (Thesis director) / Wang, Xiao (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Extrachromosomal circular DNA (eccDNA) has been identified in a broad range of eukaryotes and have been shown to carry genes and regulatory sequences. Additionally, they can amplify within a cell by autonomous replication or reintegration into the genome, effectively influencing copy number in cells. This has significant implications for cancer, where oncogenes are frequently amplified on eccDNA. However, little is known about the exact molecular mechanisms governing eccDNA functionality. To this end, we constructed a fluorescent reporter at an eccDNA-prone locus of the yeast genome, CUP1. It is our hope that this reporter will contribute to a better understanding of eccDNA formation and amplification within a cell.
ContributorsKeal, Tula Ann (Author) / Wang, Xiao (Thesis director) / Tian, Xiaojun (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Clustered regularly interspace short palindromic repeats (CRISPR) and CRISPR associated (Cas) technologies have become integral to genome editing. Canonical CRISPR-Cas9 systems function as a ribonucleic acid (RNA)-guided nucleases. Single guide RNAs (sgRNA) can be easily designed to target Cas9’s nuclease activity towards protospacer deoxyribonucleic acid (DNA) sequences. The relatively ease and efficiency of CRISPR-Cas9 systems has enabled numerous technologies and DNA manipulations. Genome engineering in human cell lines is centered around the study of genetic contribution to disease phenotypes. However, canonical CRISPR-Cas9 systems are largely reliant on double stranded DNA breaks (DSBs). DSBs can induce unintended genomic changes including deletions and complex rearrangements. Likewise, DSBs can induce apoptosis and cell cycle arrest confounding applications of Cas9-based systems for disease modeling. Base editors are a novel class of nicking Cas9 engineered with a cytidine or adenosine deaminase. Base editors can install single letter DNA edits without DSBs. However, detecting single letter DNA edits is cumbersome, requiring onerous DNA isolation and sequencing, hampering experimental throughput. This document describes the creation of a fluorescent reporter system to detect Cytosine-to-Thymine (C-to-T) base editing. The fluorescent reporter utilizes an engineered blue fluorescent protein (BFP) that is converted to green fluorescent protein (GFP) upon targeted C-to-T conversion. The BFP-to-GFP conversion enables the creation of a strategy to isolate edited cell populations, termed Transient Reporter for Editing Enrichment (TREE). TREE increases the ease of optimizing base editor designs and assists in editing cell types recalcitrant to DNA editing. More recently, Prime editing has been demonstrated to introduce user defined DNA edits without the need for DSBs and donor DNA. Prime editing requires specialized prime editing guide RNAs (pegRNAs). pegRNAs are however difficult to manually design. This document describes the creation of a software tool: Prime Induced Nucleotide Engineering Creator of New Edits (PINE-CONE). PINE-CONE rapidly designs pegRNAs based off basic edit information and will assist with synthetic biology and biomedical research.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis advisor) / Brafman, David A (Committee member) / Tian, Xiao-jun (Committee member) / Nielsen, David R (Committee member) / Arizona State University (Publisher)
Created2023
Description
The WNT signaling pathway plays numerous roles in development and maintenance of adult homeostasis. In concordance with it’s numerous roles, dysfunction of WNT signaling leads to a variety of human diseases ranging from developmental disorders to cancer. WNT signaling is composed of a family of 19 WNT soluble secreted glycoproteins, which are evolutionarily conserved across all phyla of the animal kingdom. WNT ligands interact most commonly with a family of receptors known as frizzled (FZ) receptors, composed of 10 independent genes. Specific interactions between WNT proteins and FZ receptors are not well characterized and are known to be promiscuous, Traditionally canonical WNT signaling is described as a binary system in which WNT signaling is either off or on. In the ‘off’ state, in the absence of a WNT ligand, cytoplasmic β-catenin is continuously degraded by the action of the APC/Axin/GSK-3β destruction complex. In the ‘on’ state, when WNT binds to its Frizzled (Fz) receptor and LRP coreceptor, this protein destruction complex is disrupted, allowing β-catenin to translocate into the nucleus where it interacts with the DNA-bound T cell factor/lymphoid factor (TCF/LEF) family of proteins to regulate target gene expression. However in a variety of systems in development and disease canonical WNT signaling acts in a gradient fashion, suggesting more complex regulation of β-catenin transcriptional activity. As such, the traditional ‘binary’ view of WNT signaling does not clearly explain how this graded signal is transmitted intracellularly to control concentration-dependent changes in gene expression and cell identity. I have developed an in vitro human pluripotent stem cell (hPSC)-based model that recapitulates the same in vivo developmental effects of the WNT signaling gradient on the anterior-posterior (A/P) patterning of the neural tube observed during early development. Using RNA-seq and ChIP-seq I have characterized β-catenin binding at different levels of WNT signaling and identified different classes of β-catenin peaks that bind cis-regulatory elements to influence neural cell fate. This work expands the traditional binary view of canonical WNT signaling and illuminates WNT/β-catenin activity in other developmental and diseased contexts.
ContributorsCutts, Joshua Patrick (Author) / Brafman, David A (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Nikkhah, Mehdi (Committee member) / Wang, Xiao (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2019
Description
Synthetic biology (SB) has become an important field of science focusing on designing and engineering new biological parts and systems, or re-designing existing biological systems for useful purposes. The dramatic growth of SB throughout the past two decades has not only provided us numerous achievements, but also brought us more timely and underexplored problems. In SB's entire history, mathematical modeling has always been an indispensable approach to predict the experimental outcomes, improve experimental design and obtain mechanism-understanding of the biological systems. \textit{Escherichia coli} (\textit{E. coli}) is one of the most important experimental platforms, its growth dynamics is the major research objective in this dissertation. Chapter 2 employs a reaction-diffusion model to predict the \textit{E. coli} colony growth on a semi-solid agar plate under multiple controls. In that chapter, a density-dependent diffusion model with non-monotonic growth to capture the colony's non-linear growth profile is introduced. Findings of the new model to experimental data are compared and contrasted with those from other proposed models. In addition, the cross-sectional profile of the colony are computed and compared with experimental data. \textit{E. coli} colony is also used to perform spatial patterns driven by designed gene circuits. In Chapter 3, a gene circuit (MINPAC) and its corresponding pattern formation results are presented. Specifically, a series of partial differential equation (PDE) models are developed to describe the pattern formation driven by the MINPAC circuit. Model simulations of the patterns based on different experimental conditions and numerical analysis of the models to obtain a deeper understanding of the mechanisms are performed and discussed. Mathematical analysis of the simplified models, including traveling wave analysis and local stability analysis, is also presented and used to explore the control strategies of the pattern formation. The interaction between the gene circuit and the host \textit{E. coli} may be crucial and even greatly affect the experimental outcomes. Chapter 4 focuses on the growth feedback between the circuit and the host cell under different nutrient conditions. Two ordinary differential equation (ODE) models are developed to describe such feedback with nutrient variation. Preliminary results on data fitting using both two models and the model dynamical analysis are included.
ContributorsHe, Changhan (Author) / Kuang, Yang (Thesis advisor) / Wang, Xiao (Committee member) / Kostelich, Eric (Committee member) / Tian, Xiaojun (Committee member) / Gumel, Abba (Committee member) / Arizona State University (Publisher)
Created2021
Description
Extrachromosomal circular DNA (eccDNA) has become an increasingly popular subject of study in eukaryotic cell biology due to its prevalence in human cancer. Though the literature reports a consensus regarding DNA break repair as a driver of eccDNA formation, there remains a lack of knowledge surrounding the exact mechanisms for eccDNA formation and the selective dynamics that promote their retainment in a cell or population. A central issue to studying eccDNA is the inability to distinguish between linear and circular DNA of homologous sequence. The work presented here describes an adapted eccDNA enrichment and detection assay, specifically for investigating the effects of manipulating a known eccDNA-forming locus in the budding yeast Saccharomyces cerevisiae. First, a galactose inducible GFP reporter was integrated within the copper inducible CUP1 tandem repeat locus of yeast cells. The eccDNA enrichment and detection assay was first applied to wildtype yeast to demonstrate the presence of CUP1 eccDNA in copper induced cells by qPCR. Although subsequent sequencing analysis failed to validate this result, it indicated the presence of various other known and previously un-reported eccDNA species. Finally, application of the enrichment protocol and qPCR detection assay to CUP1-GFP reporter cells yielded inconclusive results, suggesting the assay requires further optimization to sensitively detect eccDNA from this altered locus. While more work is necessary to draw conclusions regarding the limits of eccDNA production at a manipulated eccDNA-forming locus, this knowledge will lend to the potential for therapeutically targeting eccDNA at the point of de novo formation.
ContributorsKeal, Tula (Author) / Wang, Xiao (Thesis advisor) / Tian, Xiaojun (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2022