Table 1 Comparison of two-step and one-pot SHERLOCK formats

From: SHERLOCK: nucleic acid detection with CRISPR nucleases

AspectTwo-step SHERLOCKOne-pot SHERLOCK
Experimental time30–60 min15–30 min
Protocol optimizationEasy (each step can be individually optimized)Difficult (more challenging compared to two-step)
Contamination riskIncreased risk of surface contamination with pre-amplification reactionDecreased risk because of single tube reaction
QuantitationDifficult (depends on finding time point at which separation is possible)Easy (owing to real-time detection)
SensitivityZeptomolar range (single molecule per milliliter)Femto- to attomolar range (1−1,000 molecule(s) per microliter). Variability in sensitivities has been observed, which we largely attribute to the viscous RPA sample composition, which can influence Cas13 detection
Appropriate applications• End-point binary detection• Time-sensitive applications (<30 min)
• Applications with challenging sample inputs (e.g., quick extractions from unpurified samples)• Quantitative applications
• High-throughput applications
• Applications with increased contamination risk (repeated testing, large target amounts)