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.

Join this year's poster presenters in the Poster Hall during the Poster Networking Hour, Wednesday, November 1st, from 10:00 AM –11:00 AM PDT, to chat live about their posters and latest research and findings.

The accurate and timely detection of disease biomarkers at the point-of-care is essential to ensure effective treatment and prevention of diseases. CRISPR-based diagnostics (CRISPR-Dx) is revolutionizing molecular diagnosis by offering exceptional sensitivity, specificity, and simplicity. Research in our laboratory focuses on leveraging CRISPR-Dx and dynamic DNA assembly to create better analytical tools for diagnosing both human and crop diseases. In this talk, I will first introduce a plasmonic CRISPR Cas12a assay that combines the trans-cleavage activity of Cas12a and DNA functionalized gold nanoparticles for the visual, colorimetric detection of grapevine viral infections. I will then describe a molecular translator approach that expands CRISPR-Dx to protein-based biomarkers. Such CRISPR-based ultrasensitive protein assays enabled the detection of attomolar concentrations of SARS-CoV-2 antibodies in serum samples collected from vaccinated kidney transplant recipients who are shown to produce “undetectable” anti-SARS-CoV-2 using standard immunoassay. Finally, I will describe the development of a CRISPR-like assay using RNA-cleaving DNAzyme coupled with isothermal sequence and signal amplification, which demonstrated enhanced accessibility and multiplexity over CRISPR-Dx.

Molecular diagnostics based on clustered regularly interspaced short palindromic repeats (CRISPR) enzyme systems have been the subject of intense research. CRISPR-associated (Cas) enzyme assays are easily reconfigurable to different nucleic acid target sequences, highly specific, and compatible with simple kits and microfluidic components. We are conducting studies of the basic CRISPR enzyme kinetics. We discovered that the great majority of all CRISPR enzyme kinetics studies show data that are inconsistent and which grossly violate basic rules of mass conservation and rate laws. Further, CRISPR assays which do not use pre-amplification often report limits of detection (LoD) which are difficult to reconcile with CRISPR kinetic rates. This widespread inconsistency makes it difficult to assess the potential of CRISPR as a diagnostics tool. Following up on this, we quantified the kinetics of a range of CRISPR-Cas systems and demonstrated that kinetics fundamentally assay LoD. We also performed a study of CRISPR specificity to small mutations, including single-nucleotide polymorphisms. We are currently developing assays that leverage CRISPR specificity for cancer detection.