Isothermal Background-Free Nucleic Acid Quantification by a One-Pot Cas13a Assay Using Droplet Microfluidics

High sensitivity and specificity nucleic aciddetection has been achieved by the Cas13a collateral effect incombination with a separate recombinase polymerase amplification(RPA). However, these emerging methods cannot provide accuratequantification of nucleic acids because the two-step assayperformance may be compromised if the RPA and Cas13areactions are simply unified in a single step. In this work, wefirst addressed the challenges associated with enzymatic incom-patibility and the macromolecular crowding effect in the one-potassay development, making the consolidated RPA-Cas13a assay afacile and robust diagnostic tool. Next, we found that the one-potreaction cannot precisely quantify the targets at low concen-trations. Thus, by leveraging droplet microfluidics, we convertedthe one-pot assay to a digital quantification format, termed Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA). Dueto the droplet compartmentation, MEDICA greatly accelerates the reaction and enables relative detection in 10 min and the end-point quantification in 25 min. Moreover, MEDICA facilitates the droplet binarization for counting because of background-freesignals generated by trans-cleavage reporting of Cas13a. Our clinical validation highlights that CRISPR-based isothermal assays arepromising for the next generation of nucleic acid quantification methods.

Automated summary

In this study, a consolidated RPA-Cas13a assay was developed by addressing the challenges of enzymatic incompatibility and macromolecular crowding effect. The assay was further transformed into a digital quantification format, named Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), using droplet microfluidics. MEDICA enables fast and accurate quantification of nucleic acids, making it a promising method for next-generation nucleic acid quantification.