CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification

Bo Tian*, Gabriel Antonio S Minero, Jeppe Fock, Martin Dufva, Mikkel Fougt Hansen

*Corresponding author for this work

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False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.
Original languageEnglish
Article numbere30
JournalNucleic acids research
Issue number5
Number of pages7
Publication statusPublished - 2020

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