Engineering programmable nucleases for in vivo and in vitro applications
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 all-in-one assay design, enabling simultaneous detection of SARS-CoV-2 and Influenza-A with high specificity and sensitivity. The second-generation SPOT system, integrating the new assay and upgraded device, offers a user-friendly diagnostic tool that meets the requirements of the market. In the second section (Chapters 4-5), I explored the in vivo applications of programmable nucleases for genome editing. In Chapter 4, I introduced zCRISPR-Cas12a, an engineered CRISPR-Cas12a system that incorporates 2-aminoadenine (base Z) into the crRNA. This modification enhances the binding affinity between the crRNA and its complementary DNA target, resulting in a highly efficient and precise genome editing system. zCRISPR-Cas12a exhibits on-target editing efficiency comparable to that of the CRISPR-Cas9 system but with significantly reduced off-target effects in mammalian cells. Moreover, this system enables precise gene knock-in and highly efficient multiplex genome editing. As a follow-up study, Chapter 5 focuses on the application of the Z-RNA engineering strategy to other CRISPR-Cas systems and their derivatives. I extended our guide RNA engineering to Cas9, CasΦ, and base editors to investigate the broad applicability of this strategy. Additionally, I explored various delivery methods for our Z-RNA-based CRISPR-Cas system, including Z-mRNA and Z-gRNA, for potential therapeutic applications. Furthermore, I investigated the feasibility of multiplex genome editing using a single transcript. Given the generalizability of the Z-RNA engineering strategy, I anticipate that this approach can be extended to other Cas systems and associated editing tools, such as base editors, prime editors, and CRISPRa/i systems, to achieve enhanced functionalities and expanded applications.
Programmable nuclease, Diagnosis, Argonaute protein, Genome editing, CRISPR-Cas, Engineering.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign