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Synthesis of Biological and Mathematical Methods for Gene Network Control

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,…

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
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Developing safe and controllable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based therapies with design principles of synthetic biology

Description
The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot be broadly translated in clinical settings due to their unintended consequences. These consequences, such as immune reactions to CRISPR, immunogenic…

The CRISPR/Cas9 gene-editing tool is currently in clinical trials as the excitement about its therapeutic potential is exponentially growing. However, many of the developed CRISPR based genome engineering methods cannot be broadly translated in clinical settings due to their unintended consequences. These consequences, such as immune reactions to CRISPR, immunogenic adverse events following receiving of adeno-associated virus (AAV) as one of the clinically relevant delivery agents, and CRISPR off-target activity in the genome, reinforces the necessity for improving the safety of CRISPR and the gene therapy vehicles. Research into designing more advanced CRISPR systems will allow for the increased ability of editing efficiency and safety for human applications. This work 1- develops strategies for decreasing the immunogenicity of CRISPR/Cas9 system components and improving the safety of CRISPR-based gene therapies for human subjects, 2- demonstrates the utility of this system in vivo for transient repression of components of innate and adaptive immunity, and 3- examines an inducible all-in-one CRISPR-based control switch to pave the way for controllable CRISPR-based therapies.
ContributorsMoghadam, Farzaneh (Author) / Kiani, Samira (Thesis advisor) / LaBaer, Josh (Committee member) / Ebrahimkhani, Mo (Committee member) / Arizona State University (Publisher)
Created2020
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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 CRISPR bacterial immune system, this dream is becoming a powerful reality. Engineered CRISPR based transcriptional regulators have enabled researchers to…

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.
ContributorsPandelakis, Matthew (Author) / Ebrahimkhani, Mohammad (Thesis director) / Kiani, Samira (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05