<span class="paragraphSection"><div class="boxTitle">Abstract</div>Current one-pot clustered regularly interspaced short palindromic repeats diagnostics are limited by the <span style="font-style: italic;">cis</span>-cleavage activity of Cas nucleases, which leads to amplicon degradation during amplification. Here, we report a streamlined strategy that overcomes this limitation. By integrating a bipartite split-crRNA into Cas12a (SCas12a), we separate target recognition from PAM dependency and completely eliminate <span style="font-style: italic;">cis</span>-cleavage while preserving robust <span style="font-style: italic;">trans</span>-cleavage. This strategy is broadly applicable for one-pot testing, compatible with recombinase polymerase amplification, RT–RPA, and loop-mediated isothermal amplification, as well as multiple Cas12a orthologs, including As, Lb, and Ct Cas12a. Moreover, the SCas12a accelerates one-pot testing with 100–1000-fold improved sensitivity and achieves &gt;10-fold reduction in time-to-signal, enabling detection of targets at attomolar levels within 30 min. Additionally, it provides single-base resolution with up to 91-fold selectivity. The system has been successfully applied to detect HPV16, SARS-CoV-2, and TP53 SNPs in clinical samples. Together, we have developed a PAM-independent and <span style="font-style: italic;">cis</span>-cleavage-free one-pot Cas12a assay, which holds strong potential for point-of-care diagnostics.</span>