Engineering programmable nucleases for in vivo and in vitro applications
type: CreativeWork
description: Programmable nucleases are powerful molecular tools that have significantly advanced biotechnology development. Enzymes such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) associated nucleases, and Argonaute nucleases (Ago) have revolutionized genome engineering by allowing precise and easy manipulation of genetic material. These reprogrammable nucleases hold immense potential not only for genome editing but also for nucleic acid detection, offering highly specific and sensitive diagnostic tools. The primary focus of my dissertation is to engineer these programmable nucleases for both in vitro and in vivo applications using synthetic biology approaches. In the first section (Chapters 2-3), I dedicated my efforts to developing a diagnostic platform based on Argonaute nucleases, called the SPOT (Scalable and Portable Testing) system. This system combines a rapid, highly sensitive, and accurate assay with a battery-powered portable device for COVID-19 diagnosis. It enables the multiplexed detection of the N gene and E gene of the SARS-CoV-2 virus in saliva samples spiked with the virus, with a limit of detection (LoD) of 0.44 copies/μL and 1.09 copies/μL, respectively, within 30 minutes. Furthermore, the SPOT system successfully analyzed 104 clinical saliva samples, exhibiting a sensitivity of 93.3% (28 out of 30 SARS-CoV-2 positive samples correctly identified) and a specificity of 98.6% (73 out of 74 SARS-CoV-2 negative samples correctly identified). This combination of speed, accuracy, sensitivity, and portability provides an accessible and cost-effective solution for COVID-19 testing in urgent settings. To simplify the overall process in terms of manufacturing and testing, I have developed and optimized a next-generation SPOT system, featuring an improved assay and an upgraded device. The new assay includes rapid room temperature sample pretreatment and an…
abstract: Programmable nucleases are powerful molecular tools that have significantly advanced biotechnology development. Enzymes such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) associated nucleases, and Argonaute nucleases (Ago) have revolutionized genome engineering by allowing precise and easy manipulation of genetic material. These reprogrammable nucleases hold immense potential not only for genome editing but also for nucleic acid detection, offering highly specific and sensitive diagnostic tools. The primary focus of my dissertation is to engineer these programmable nucleases for both in vitro and in vivo applications using synthetic biology approaches. In the first section (Chapters 2-3), I dedicated my efforts to developing a diagnostic platform based on Argonaute nucleases, called the SPOT (Scalable and Portable Testing) system. This system combines a rapid, highly sensitive, and accurate assay with a battery-powered portable device for COVID-19 diagnosis. It enables the multiplexed detection of the N gene and E gene of the SARS-CoV-2 virus in saliva samples spiked with the virus, with a limit of detection (LoD) of 0.44 copies/μL and 1.09 copies/μL, respectively, within 30 minutes. Furthermore, the SPOT system successfully analyzed 104 clinical saliva samples, exhibiting a sensitivity of 93.3% (28 out of 30 SARS-CoV-2 positive samples correctly identified) and a specificity of 98.6% (73 out of 74 SARS-CoV-2 negative samples correctly identified). This combination of speed, accuracy, sensitivity, and portability provides an accessible and cost-effective solution for COVID-19 testing in urgent settings. To simplify the overall process in terms of manufacturing and testing, I have developed and optimized a next-generation SPOT system, featuring an improved assay and an upgraded device. The new assay includes rapid room temperature sample pretreatment and an…
author: Xun, Guanhua
language: en
keywords: Programmable nuclease, Diagnosis, Argonaute protein, Genome editing, CRISPR-Cas, Engineering.
date published: 2024-08
date modified: 2026-07-22T22:25:02Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: https://hdl.handle.net/2142/125741