RNA-Activated CRISPR/Cas12a Nanorobots Operating in Living Cells

Active clustered regularly interspaced short palindromic repeats (CRISPR/Cas12a) systems possess both <i>cis</i>-cleavage (targeted) and <i>trans</i>-cleavage (collateral) activities, which are useful for genome engineering and diagnostic applications. Both single- and double-stranded DNA can activate crRNA-Cas12a ribonucleoprotein (RNP) to achieve <i>cis</i>- and <i>trans</i>-cleavage enzymatic activities. However, it is not clear whether RNA can activate the CRISPR/Cas12a system and what is critical to the <i>trans</i>-cleavage activity. We report here that RNA can activate the CRISPR/Cas12a system and trigger its <i>trans</i>-cleavage activity. We reveal that the activated crRNA-Cas12a RNP favors the <i>trans</i>-cleavage of longer sequences than commonly used. These new findings of the RNA-activated <i>trans</i>-cleavage capability of Cas12a provided the foundation for the design and construction of CRISPR nanorobots that operate in living cells. We assembled the crRNA-Cas12a RNP and nucleic acid substrates on gold nanoparticles to form CRISPR nanorobots, which dramatically increased the local effective concentration of the substrate in relation to the RNP and the <i>trans</i>-cleavage kinetics. Binding of the target microRNA to the crRNA-Cas12a RNP activated the nanorobots and their <i>trans</i>-cleavage function. The repeated (multiple-turnover) <i>trans</i>-cleavage of the fluorophore-labeled substrates generated amplified fluorescence signals. Sensitive and real-time imaging of specific microRNA in live cells demonstrated the promising potential of the CRISPR nanorobot system for future applications in monitoring and modulating biological functions within living cells.