Thesis advisor: Jia Niu / In the last ten years, programmable CRISPR-Cas systems have been widely-used as genome editing tools for gene manipulation, epigenetic functionalization, and transcriptional regulation. Among them, fusing effector proteins directly to the Cas protein allows the resulting CRISPR machinery to direct these effector proteins to multiple sites of the same gene or multiple genes at once. Although they can be used to target multiple genetic loci simultaneously, these methods are often limited to applying one regulatory function (e.g., activation or repression) at a time. On the other hand, recruiting effector proteins via RNA aptamer-RNA-binding protein (RBP) recognition enabled multiplexed and multi-modular gene manipulations simultaneously. However, there are only a limited set of aptamer-RBP pairs that can function orthogonally and intracellularly, e.g., MS2 RNA aptamer with MS2 coat protein (MCP), and PP7 RNA aptamer with PP7 coat protein (PCP). The scarcity of orthogonal intracellular aptamer-RBP pairs imposes severe constraints on the CRISPR-mediated multifunctional manipulations of the genome and the epigenome. We established an intracellular selection platform for RNA aptamers, named CRISPR-Hybrid, and expanded the scope of aptamer-RBP toolkit for CRISPR transcription regulators. Using CRISPR-Hybrid, we successfully identified a highly active and specific aptamer for bacteriophage Qβ coat protein (QCP) in vivo, and characterized its binding affinity and specificity in vitro. We further validated the orthogonality of selected aptamer with QCP to other available intracellularly functional aptamer-RBP pairs including MS2-MCP and PP7-PCP in mammalian cells. Finally, we demonstrated the utility of this orthogonal pair in multiplexed and multi-modular regulations of endogenous genes. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
| Identifer | oai:union.ndltd.org:BOSTON/oai:dlib.bc.edu:bc-ir_110048 |
| Date | January 2024 |
| Creators | Su-Tobon, Qiwen |
| Publisher | Boston College |
| Source Sets | Boston College |
| Language | English |
| Detected Language | English |
| Type | Text, thesis |
| Format | electronic, application/pdf |
| Rights | Copyright is held by the author, with all rights reserved, unless otherwise noted. |
Page generated in 0.002 seconds