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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
Synthetic biology is an emerging field which melds genetics, molecular biology, network theory, and mathematical systems to understand, build, and predict gene network behavior. As an engineering discipline, developing a mathematical understanding of the genetic circuits being studied is of fundamental importance. In this dissertation, mathematical concepts for understanding, predicting, and controlling gene transcriptional networks are presented and applied to two synthetic gene network contexts. First, this engineering approach is used to improve the function of the guide ribonucleic acid (gRNA)-targeted, dCas9-regulated transcriptional cascades through analysis and targeted modification of the RNA transcript. In so doing, a fluorescent guide RNA (fgRNA) is developed to more clearly observe gRNA dynamics and aid design. It is shown that through careful optimization, RNA Polymerase II (Pol II) driven gRNA transcripts can be strong enough to exhibit measurable cascading behavior, previously only shown in RNA Polymerase III (Pol III) circuits. Second, inherent gene expression noise is used to achieve precise fractional differentiation of a population. Mathematical methods are employed to predict and understand the observed behavior, and metrics for analyzing and quantifying similar differentiation kinetics are presented. Through careful mathematical analysis and simulation, coupled with experimental data, two methods for achieving ratio control are presented, with the optimal schema for any application being dependent on the noisiness of the system under study. Together, these studies push the boundaries of gene network control, with potential applications in stem cell differentiation, therapeutics, and bio-production.
ContributorsMenn, David J (Author) / Wang, Xiao (Thesis advisor) / Kiani, Samira (Committee member) / Haynes, Karmella (Committee member) / Nielsen, David (Committee member) / Marshall, Pamela (Committee member) / Arizona State University (Publisher)
Created2018
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
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.
ContributorsPineda, Michael (Author) / Kiani, Samira (Thesis director) / Ebrahimkhani, Mo (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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 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.
ContributorsStandage-Beier, Kylie S (Author) / Wang, Xiao (Thesis director) / Haynes, Karmella (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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 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
ContributorsSacco, Elena Maria (Author) / Frow, Emma (Thesis director) / Maynard, Andrew (Committee member) / School of Politics and Global Studies (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Synthetic biology is a novel method that reengineers functional parts of natural genes of interest to build new biomolecular devices able to express as designed. There is increasing interest in synthetic biology due to wide potential applications in various fields such as clinics and fuel production. However, there are still many challenges in synthetic biology. For example, many natural biological processes are poorly understood, and these could be more thoroughly studied through model synthetic gene networks. Additionally, since synthetic biology applications may have numerous design constraints, more inducer systems should be developed to satisfy different requirements for genetic design.
This thesis covers two topics. First, I attempt to generate stochastic resonance (SR) in a biological system. Synthetic bistable systems were chosen because the inducer range in which they exhibit bistability can satisfy one of the three requirements of SR: a weak periodic force is unable to make the transition between states happen. I synthesized several different bistable systems, including toggle switches and self-activators, to select systems matching another requirement: the system has a clear threshold between the two energy states. Their bistability was verified and characterized. At the same time, I attempted to figure out the third requirement for SR – an effective noise serving as the stochastic force – through one of the most widespread toggles, the mutual inhibition toggle, in both yeast and E. coli. A mathematic model for SR was written and adjusted.
Secondly, I began work on designing a new genetic system capable of responding to pulsed magnetic fields. The operators responding to pulsed magnetic stimuli in the rpoH promoter were extracted and reorganized. Different versions of the rpoH promoter were generated and tested, and their varying responsiveness to magnetic fields was recorded. In order to improve efficiency and produce better operators, a directed evolution method was applied with the help of a CRISPR-dCas9 nicking system. The best performing promoters thus far show a five-fold difference in gene expression between trials with and without the magnetic field.
This thesis covers two topics. First, I attempt to generate stochastic resonance (SR) in a biological system. Synthetic bistable systems were chosen because the inducer range in which they exhibit bistability can satisfy one of the three requirements of SR: a weak periodic force is unable to make the transition between states happen. I synthesized several different bistable systems, including toggle switches and self-activators, to select systems matching another requirement: the system has a clear threshold between the two energy states. Their bistability was verified and characterized. At the same time, I attempted to figure out the third requirement for SR – an effective noise serving as the stochastic force – through one of the most widespread toggles, the mutual inhibition toggle, in both yeast and E. coli. A mathematic model for SR was written and adjusted.
Secondly, I began work on designing a new genetic system capable of responding to pulsed magnetic fields. The operators responding to pulsed magnetic stimuli in the rpoH promoter were extracted and reorganized. Different versions of the rpoH promoter were generated and tested, and their varying responsiveness to magnetic fields was recorded. In order to improve efficiency and produce better operators, a directed evolution method was applied with the help of a CRISPR-dCas9 nicking system. The best performing promoters thus far show a five-fold difference in gene expression between trials with and without the magnetic field.
ContributorsHu, Hao (Author) / Wang, Xiao (Thesis advisor) / Stabenfeldt, Sarah (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2016
ContributorsEvans, Bartlett R. (Conductor) / Schildkret, David (Conductor) / Glenn, Erica (Conductor) / Concert Choir (Performer) / Chamber Singers (Performer) / ASU Library. Music Library (Publisher)
Created2018-03-16
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 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.
ContributorsLakers, Mary Frances (Author) / Brafman, David (Thesis advisor) / Haynes, Karmella (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2017