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Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.<\/jats:p>","DOI":"10.1093\/nar\/gkag167","type":"journal-article","created":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T12:41:51Z","timestamp":1771418511000},"source":"Crossref","is-referenced-by-count":7,"title":["crRNA scaffold remodeling controls CRISPR\u2013Cas12a activity for enhanced performance"],"prefix":"10.1093","volume":"54","author":[{"given":"Zhongzhong","family":"Wang","sequence":"first","affiliation":[{"name":"Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital Medical University , Beijing 100069 ,","place":["China"]},{"name":"Beijing Key Laboratory of Environment and Aging , Beijing 100069 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