Recombinase Polymerase Amplification (RPA) coupled with CRISPR-based detection offers a rapid, highly sensitive, and isothermal molecular diagnostic framework, bypassing the need for complex thermal cyclers. Operating at a constant 37°C to 42°C, the workflow amplifies target DNA or RNA in 10 to 30 minutes before introducing a sequence-specific guide RNA (sgRNA) and CRISPR nuclease to trigger collateral cleavage of a signal-generating reporter probe.

This protocol outlines a flexible, multi-format approach to execute and optimize RPA-CRISPR diagnostic assays directly from sample targets.  This protocol details three execution formats to suit different development stages and target types:

  • Two-Pot (Sequential) Development Workflow: Amplification and CRISPR detection occur in separate tubes, allowing independent optimization of reaction kinetics while preventing carryover inhibition.
  • One-Pot (Homogeneous) Setup: Combines amplification and CRISPR detection into a single closed tube to reduce hands-on time once buffer compatibility and enzyme ratios are established.
  • Cas13 Adaptations: Incorporates a T7-mediated in vitro transcription (IVT) step and ssRNA probes to enable direct or transcribed RNA target detection.

By eliminating complex cycling equipment and offering flexible, modular execution paths, this workflow provides an adaptable blueprint for accelerating point-of-care diagnostic development. Explore our RPA and RT-RPA kits.

For a comprehensive, high-level approach to planning your entire experimental design before diving into the execution guide, please reference our 9-Step Guide to Building an RPA-CRISPR Diagnostic Assay.