Applying CRISPR/Cas13 to Construct Exosomal PD‐L1 Ultrasensitive Biosensors for Dynamic Monitoring of Tumor Progression in Immunotherapy

Abstract

Programmed cell death receptor 1 (PD-L1) protein on exosomes (exosomal PD-L1) is one of the most promising biomarkers for cancer immunotherapy monitoring. However, current approaches for exosomal PD-L1 detection are poorly sensitive, laborious, and time-consuming. Here, a new method, named Aptamer-RPA-TMA-Cas13a Assay (ARTCA) is established, which enables exosomal PD-L1 to be detected directly in serum with a lower limit of 10 particles mL −1. Mechanistically, using DNA aptamer specifically binding to exosomal PD-L1, the aptamer is amplified twice by recombinase polymerase amplification (RPA) coupled with transcription-mediated amplification (TMA) and simultaneously the TMA products are detected in real-time with CRISPR/Cas13a system. Utilizing ARTCA, PD-L1 levels in circulating exosomes seem to be a reliable marker of PD-L1 expression in tumor tissue. The level of circulating exosomal PD-L1 increases significantly in patients with tumor progression. Ultra-trace detection of serum exosomal PD-L1 by ARTCA provides a potentially convenient way for dynamic monitoring of tumor progression for patients undergoing immunotherapy. These results demonstrate the use of CRISPR-Cas13a for protein detection, and circulating exosomal PD-L1 levels seem to be a reliable marker as well as PD-L1 expression in tumor tissue, opening up new avenues for monitoring tumor progression.

Advances in Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-Based Diagnostic Assays Assisted by Micro/Nanotechnologies

“…Currently, CRISPR biosensors are no longer limited to nucleic acid analyses. The robust signal amplification mechanism of CRISPR-Cas12 and CRISPR-Cas13 makes it a promising tool for sensing non-nucleic-acid targets, such as pathogenic bacteria, exosomes, − small molecules, ,− proteins, − and ions . Unlike nucleic acid detection, which can achieve ultrahigh sensitivity through template amplification, there is currently no viable non-nucleic-acid target amplification method, so its detection sensitivity conventionally depends on the probe affinity and detector performance.…”

CRISPR/Cas-Based In Vitro Diagnostic Platforms for Cancer Biomarker Detection

“…The amplicon of RPA further initiated the transcription-mediated amplification (TMA) to acquire the RNA transcripts to activate CRISPR/ Cas13a. 113 The LOD was determined to be 10 particles/mL, and the strategy showed promise in dynamically monitoring the progression in immunotherapy.…”

“…The PD-L1 on exosomes was bound with PD-L1 aptamers, which could be finally eluted and serve as the trigger for RPA amplification. The amplicon of RPA further initiated the transcription-mediated amplification (TMA) to acquire the RNA transcripts to activate CRISPR/Cas13a . The LOD was determined to be 10 particles/mL, and the strategy showed promise in dynamically monitoring the progression in immunotherapy.…”

CRISPR-Cas13 System as a Promising and Versatile Tool for Cancer Diagnosis, Therapy, and Research

“…New protein analysis methods such as bead-based flow cytometry and nanoplasmonic exosome sensor have been developed to detect exosomal proteins; however, they are expensive and still requires special equipment . To circumvent these restrictions, He et al developed a biosensor for exosomal protein detection by combining DNA aptamer-based protein recognition, RPA, and Cas13-based signal augmentation . They tested the biosensor to detect serum exosomal programmed death-ligand 1 (PD-L1), which is a potential marker for immunotherapy monitoring.…”

“…155 To circumvent these restrictions, He et al developed a biosensor for exosomal protein detection by combining DNA aptamer-based protein recognition, RPA, and Cas13-based signal augmentation. 100 They tested the biosensor to detect serum exosomal programmed death-ligand 1 (PD-L1), which is a potential marker for immunotherapy monitoring. In this method, exosomes are captured from patients' blood using an antibody cocktail.…”