Keynote Presentation: Microfluidics and CRISPR-Diagnostics/Therapy with Live Q&A

Coronavirus Disease 2019 (COVID-19) has driven the use of nucleic acid testing technology in clinical testing. There is a growing need for point-of-care testing (POCT) devices that can integrate sample pretreatment and nucleic acid detection in a rapid, cost-effective, and non-labor-intensive manner. The most widely used polymerase chain reaction (PCR)-based nucleic acid assays suffer from complexity, high personnel and laboratory requirements, and contamination. Highly miniaturized microfluidic chips provide the necessary tools to integrate complex nucleic acid detection processes. Various microfluidic chips coupled with clustered regularly interspaced short palindromic repeat (CRISPR) have been developed for the rapid detection of nucleic acids. These tools offer a range of advantages including high sensitivity, high automation, high specificity, and a high level of integration performance, and we have developed a series of nucleic acid detection systems based on CRISPR and microfluidic technologies:

(1) High sensitivity. An integrated assay for nucleic acid enrichment and single-molecule digital detection based on CRISPR/Cas13a and microwell arrays. The design utilizes magnetic beads to capture and enrich targets from a large number of samples, and then target-induced CRISPR/Cas13a cleavage reactions are dispersed and confined to 1 million femtoliter-sized microwells to enhance local signal intensity for single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. This "sample-in-answer-out" amplification-free single RNA detection technology improves sensitivity and specificity while reducing detection time.

(2) High automation, rapid detection. The dual CRISPR/Cas12a-assisted RT- recombinase-assisted amplification (RAA) assay and "sample-to-answer" centrifugal microfluidic platform automatically detects 1 copy/μL of SARS-CoV-2 in less than 30 minutes. The chip separates the amplification (RAA) from the detection (CRISPR) to maximize sensitivity and time consumption. The chip separates amplification (RAA) from detection (CRISPR) to maximize sensitivity and reduce time consumption by up to 3-fold. This automated centrifugal microfluidic technology achieved 100% accuracy on 34 clinical SARS-CoV-2 samples compared to the gold standard RT-PCR technique.

(3）High specificity. A universal and high-fidelity genotyping method based on a microfluidic point-of-care device for CRISPR systems. The versatility of the CRISPR / Cas12a-based SNP assay was improved by systematically inserting protospacer adjacent motif (PAM) sequences, and the sensitivity and specificity were improved by removing complementary ssDNA and introducing other nucleotide mismatches.

(4) High integration performance. A centrifugal microfluidic platform that allows for the separation of serum as well as a range of functions such as adsorption, washing, elution, and detection of DNA. The platform combines a variety of signal enhancement systems, including recombinase polymerase amplification (RPA), T7 transcription, and CRISPR, to generate ultra-bright signals, and hepatitis B virus (HBV) has been successfully detected and genotyped from whole blood on an automated centrifugal microfluidic platform. The automated centrifugal microfluidic platform with integrated preprocessing and molecular diagnostics play an important role in clinical testing.

I will also briefly discuss our effort in integrating CRISPR-based therapies with nanomaterials at the end of the talk.

Learning Objectives:

1. Summarize CRISPR/Cas12a-Assisted RT-RAA for Ultrasensitive SARS-CoV-2 Detection.

2. Explain RPA-T7-Cas13a for pathogen diagnosis in 30 min. 

3. Review CRISPR/Cas-assisted assay for single molecule detection of RNA with out amplification on chip.