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Improving One-Pot RPA−CRISPR-Cas12 Assays

CRISPR-Cas systems are potent tools for detecting nucleic acid sequences, especially when combined with nucleic acid amplification techniques (NAATs) such as recombinase polymerase amplification (RPA). Highest diagnostic sensitivities are normally achieved when nucleic acid amplification and CRISPR-Cas detection are performed sequentially. However, sequential methods involve multiple steps, are difficult to automate and prone to contamination, limiting application in point-of-care scenarios. To address this issue, “one-pot” formats integrate both processes in a single step. Whilst being simple to implement, one-pot methods often suffer from reduced sensitivity due to the fact that Cas protein exonuclease activity interferes with nucleic acid amplification.

Jake M. Lesinski and colleagues from ETH Zurich and Ghent University have recently shown that high sensitivity one-pot assays can in fact be realized by controlling Cas-based ribonucleoprotein (RNP) concentration at the beginning of the assay to balance exonuclease activity and amplification. Their method simplifies workflows and ensures high sensitivities without the need for complex workflows. By comparing two one-pot RPA–CRISPR-Cas12a assay models the team found that the in-situ assay could detect lower target concentrations (0.2 copies/μL) that the precomplexed assay (2 copies/μL). This observation supports the hypothesis that the cis exonuclease activity of the Cas protein hinders DNA amplification at low DNA concentrations. Subsequently, the authors tested robustness of the in situ complexed assay using clinical samples. Encouragingly, data show that the in-situ assay more closely aligns with the Allplex qPCR data than the precomplexed assay. Finally, the team investigated the mechanisms behind the improved limit of detection and sensitivity for the in situ one-pot RPA–Cas12a assay. In summary, the team have shown that robust detection of low-titer targets is possible when using one-pot protocols provided that cis exonuclease activity can be minimized during early assay times.

Written by Chao Song

Read the published article here.

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