Associate Professor University of Connecticut Health Center, United States
Introduction:: The COVID-19 pandemic highlighted the importance of accurate and rapid nucleic acid detection in clinical diagnostics. While PCR is the gold standard method, alternative methods are needed for rapid, cost-effective, and easy-to-use diagnostic tools. Recently, the CRISPR-Cas system, originally a bacterial immune system, has been repurposed for nucleic acid detection. Various CRISPR-based methods have been developed to date and involve pre-amplification of target nucleic acids, followed by detection using Cas enzymes, generating fluorescent, colorimetric, or electrochemical readouts. Cas12a is a programmable DNA endonuclease for gene editing and molecular diagnostics. Cas12a has trans-cleavage activity and is widely applied to detect dsDNA or ssDNA, but additional manipulation is required for RNA detection. Recently, efforts have been made to improve the sensitivity and specificity by improving the catalytic efficiency of Cas12a using crRNA modifications. In addition, it has been reported that split crRNAs containing separated scaffold and spacer RNAs can catalyze highly specific and efficient cleavage of target DNA via Cas12a. However, its effect on trans-cleavage is unknown.
Materials and Methods:: The competitive crRNA reaction-based cascade signal amplification method uses target-specific full-sized crRNA and independent split crRNA and its target ssDNA (split-T) to enhance sensitivity. The full-sized crRNA is designed to bind to target nucleic acids (full-T), and the split crRNA and split-T are separately designed. The reaction solution was prepared by mixing the Cas12a, a fluorescence reporter dye (5’-FAM-TTATT-Quencher-3’), full-sized crRNA, split crRNA, and split-T. The real-time fluorescence signal was measured after mixing the reaction solution with different concentrations of full-T.
Results, Conclusions, and Discussions:: We demonstrated the full-sized crRNA had a stronger binding affinity to Cas12a than the split crRNA. As a result, under conditions where both full-sized and split crRNA were mixed, we observed a more dominant formation of the Cas12a/full-sized crRNA complex over the Cas12a/split crRNA complex. Therefore, when two different types of crRNA and split crRNA target (split-T) were mixed, the trans-cleavage reaction of split crRNA and split-T was inhibited due to the asymmetric Cas12a/crRNA complex formation. Then, once the full-T is added, the trans-cleavage activity is induced by Cas12a/full-sized crRNA first, and full-sized crRNA is consumed through the Cas12a/crRNA kinetics. Subsequently, the split crRNA reaction can be activated, resulting in additional fluorescence signal amplification. Based on this, we completed the asymmetric CRISPR assay that can achieve highly sensitive target nucleic acid detection using independent split crRNAs and split-T sets. (Scheme 1)
In this study, we have revealed interesting CRISPR/Cas12a activities, including its ability to recognize split targets and competitive crRNA dynamics, which can be leveraged for signal amplification and miRNA detection. We have demonstrated that Cas12a can directly recognize short ssRNA when supported by ssDNA located at the 5’ end of crRNA. Moreover, we have shown that full-sized crRNA has a stronger binding affinity to Cas12a than split crRNA, allowing for a competitive crRNA cascade reaction that improves the detection sensitivity of full-sized crRNA. Based on these findings, we developed a one-pot, one-step isothermal signal amplification method using one enzyme (Cas12a) capable of detecting the attomolar level of miRNA, which can also be applied to DNA detection. By using the developed assay, we could successfully analyze one of the cancer biomarkers, miR-19a, in the bladder cancer patient samples. We believe that our findings prove a valuable contribution to the field of CRISPR-based diagnostics and liquid biopsy. Overall, our study highlights the versatility and potential of the CRISPR/Cas12a system for nucleic acid detection and presents a new approach for miRNA detection that could have important clinical applications.
