<span class="paragraphSection"><div class="boxTitle">Abstract</div>One-pot CRISPR-based diagnostics have transformed nucleic acid testing, yet their design customizability remains constrained. Because target programming and <span style="font-style: italic;">cis-</span>cleavage activity are simultaneously determined during CRISPR RNA (crRNA) design, optimizing cleavage activity to match isothermal amplification inevitably requires altering the programmed crRNA sequence. This requirement fundamentally constrains the range of compatible target sequences, imposing limitations on the flexible design of diagnostic assays. Here, we establish a customizable one-pot system by decoupling the dual functions inherent in crRNA design to enable their independent control. In this strategy, target programming remains defined by the crRNA sequence, whereas <span style="font-style: italic;">cis-</span>cleavage activity is regulated by the reaction energy barrier. We selectively modulate this energy barrier through the introduction of a crRNA–complementary RNA oligonucleotide, achieving cleavage regulation without altering the crRNA sequence. Consequently, this approach ensures that <span style="font-style: italic;">cis-</span>cleavage activity matches isothermal amplification conditions independent of the programmed target sequence, thereby realizing a customizable CRISPR diagnostic system. We validated the clinical applicability of this system using 120 patient-derived samples, achieving sensitivity and specificity comparable to quantitative polymerase chain reaction. Collectively, this work resolves a fundamental constraint of CRISPR diagnostics and establishes a customizable and clinically deployable platform for next-generation nucleic acid testing.</span>