CRISPR/Cas12a-Modulated fluorescence resonance energy transfer with nanomaterials for nucleic acid sensing

摘要

Cas12a shows great promise in DNA sensing applications due to its target-triggered collateral trans-cleavage activity. But the cleavage effect towards probes modified on nanomaterials is less understood. In this work, an analogy analysis is performed to explore the cleavage properties of Cas12a system on the surface of nanomaterials by using gold nanoparticles (AuNPs) and graphene oxide (GO). The fluorescence of FAM-tagged probes is quenched by nanomaterials due to fluorescence resonance energy transfer (FRET). The (ram-cleavage activity of Cas12a was activated to promiscuously digest the single-stranded part of probes, thereby modulating FRET effect between FAM labels and nanomaterials. The results showed that GO had better quenching ability and Cas12a-induced fluorescence recovery than AuNPs, and the double-stranded DNA probe with staggered end can lead preferable trans-cleavage efficiency, proving low steric hindrance of nanomaterials and appropriate orientation of probes play critical roles in Cas12a-modulated FRET with nanomaterials. Then, by integrating Cas12a system and GO-based post-quenching strategy, a DNA biosensor was developed with extremely high fluorescence response and low initial background. It is expected that this work will be of particularly useful for investigation of the interaction between CRISPR/Cas proteins and nanomaterials, as well as developing CRISPR-based point-of-care (POC) diagnostic platforms.