The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for simple, low-cost, and scalable diagnostics that can be widely deployed for rapid testing.Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as a promising technol., but its implementation in clin. laboratories has been limited by the requirement of a sep. amplification step prior to CRISPR-associated (Cas) enzyme-based detection.This article reports the discovery of two novel Cas12 enzymes [STE20-like serine/threonine-protein kinase (SLK)-9 and SLK5-2] that exhibit enzymic activity at 60°C, which, when combined with loop-mediated isothermal amplification, enable a real-time, single-step nucleic acid detection method (termed real-time SLK, pronounced Sherlock).Real-time SLK was demonstrated to provide accurate results comparable to those from real-time quant. RT-PCR in clin. samples, with 100% pos. and 100% neg. percent agreement.The method is further demonstrated to be compatible with direct testing (real-time SLK Direct) of samples from anterior nasal swabs, without the need for standard nucleic acid extractionLastly, SLK9 was combined with either Alicyclobacillus acidoterrestris (Aac)-Cas12b or with SLK5-2 to generate a real-time, multiplexed CRISPR-based diagnostic assay for the simultaneous detection of SARS-CoV-2 and a human-based control in a single reaction, with sensitivity down to 5 copies/μL and a time to result of under 30 min.