A Rational Design Framework for Generalizable and Modular One-pot CRISPR Diagnostics

Abstract

CRISPR-based one-pot nucleic acid diagnostics are recognized as a next-generation approach for nucleic acid detection because they extend testing beyond centralized laboratories to decentralized and point-of-care settings. However, this approach has not yet been established as a generalizable implementation principle comparable to qPCR, thereby limiting its broad applicability and scalability. In this dissertation, we develop a generalizable one- pot CRISPR design framework. This framework is further extended into a modular one-pot platform that is compatible with diverse isothermal amplification chemistries and applicable across different target contexts. Chapter 1 provides the general background of this dissertation, beginning with the biological and clinical significance of nucleic acid information and the role of nucleic acid testing in modern diagnostics. It then introduces qPCR as the current gold standard and discusses the paradigm shift toward decentralized nucleic acid testing. Against this background, CRISPR-based diagnostics are presented as an emerging strategy for next- generation nucleic acid detection. Their development is then reviewed from trans-cleavage-based detection to two-step and one-pot formats, highlighting the remaining challenge of establishing broadly applicable implementation principles for one-pot CRISPR systems. Chapter 2 describes the development of a generalizable one-pot CRISPR design framework that overcomes a fundamental limitation of conventional simultaneous amplification-cleavage strategies. In conventional one-pot CRISPR systems, target programming and cleavage behavior are tightly coupled to the programmed sequence, thereby limiting sequence-level customizability. To address this constraint, an RNA oligonucleotide complementary to the crRNA spacer is introduced to form an artificial secondary structure and modulate the free- energy barrier governing cis-cleavage activity. This enables predictable length-dependent control of cleavage behavior and allows one-pot CRISPR reactions to be implemented through a rational design strategy without altering the programmed target sequence. Chapter 3 presents the extension and application of this generalizable design framework toward temperature- universal and modular one-pot CRISPR implementation. By replacing RNA with LNA and tuning its substitution density, the same design principle is extended to control reaction behavior across a wider temperature range. This temperature-universal expansion enables compatibility with multiple isothermal amplification chemistries that operate under different thermal conditions. As a result, the framework becomes adaptable to diverse target contexts in which different amplification chemistries are required. Its applicability is further demonstrated through diagnostic applications. Taken together, this dissertation establishes a generalizable design framework for one-pot CRISPR systems, extends it toward temperature-universal and modular implementation, and applies it across diverse target contexts. By providing a broadly applicable conceptual and practical basis, this work is expected to facilitate the development of diagnostics for diverse targets and support the broader translation of CRISPR-based nucleic acid testing from centralized laboratories to decentralized and point-of-care settings.