Integration of PNA-mediated PCR and CRISPR/Cas13a for highly sensitive detection of EGFR T790M mutation in circulating tumor DNA.

One-pot SP-EXPAR-integrated CRISPR/Cas14a assay for highly specific and versatile detection of ctDNA mutations in lung cancer patients.

A one-pot self-primer isothermal exponential amplification reaction (SP-EXPAR) combined with a CRISPR/Cas14a assay was developed for detecting KRAS G12C and G12D and demonstrated 100% sensitivity and 100% specificity compared with DNA sequencing.

Analytical validation of a modified wild-type blocker qpcr assay for the sensitive DNA-based detection of NPM1 type A in acute myeloid leukemia

5′ Rapid Amplification of cDNA Ends (5′ RACE)-Based Targeted RNA Sequencing Assay for the Analysis of Clinically Relevant Alterations in Lung Cancer Patients

Next-generation sequencing (NGS) is currently regarded as a preferable method for detection of actionable alterations in non-small cell lung carcinomas (NSCLCs). RNA-based NGS is particularly efficient in detection of gene fusions and allows for mRNA expression analysis of relevant genes. This study describes a novel library preparation pipeline for targeted RNA sequencing, which is based on the 5′ rapid amplification of the cDNA ends (5′ RACE) and anchored multiplex PCR. The NGS panel was designed for the analysis of 15 genes relevant to NSCLC therapeutic decisions (ALK, BRAF, CD274 (PD-L1), EGFR, ERBB2 (HER2), KRAS, MET, NRAS, NRG1-2, NTRK1-3, RET and ROS1). The validation against PCR was performed for 168 NSCLC samples. The NGS assay successfully detected all 85 mutations previously identified by PCR. It also confirmed the absence of tested mutations in 82 out of 83 PCR-negative samples. The only discordant case was subsequently analyzed by digital droplet PCR and demonstrated low fraction of the mutated allele. The newly designed NGS panel was subsequently used for the analysis of 272 carcinomas from young (≤50 years old) patients with no driver mutations identified by PCR tests or with failed PCR analysis. NGS was successful in 240 (88.2%) cases, including 35 (64.8%) PCR-failed samples. Driver mutations were detected in 55 tumors, including three previously unreported fusions (PAPLN::ALK, CD55::ROS1 and FKBP15::RET). We conclude that 5′ RACE-based RNA sequencing is a viable approach for NSCLC molecular testing.

Signal logic gate mediated by a controllable CRISPR/Cas12a system for simultaneous detection of DNA mutation and methylation.

A controllable CRISPR/Cas12a-based signal logic gate that enables simultaneous detection of KRAS G12C mutation and Septin9 promoter methylation in a single reaction and offers a simple and reliable platform for simultaneous genetic and epigenetic analysis in point-of-care cancer screening.

Universal CRISPR/Cas12a-G4 DNAzyme biosensing platform for rapid detection of nucleic acid and non-nucleic acid targets.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system enables sensitive and specific biomolecular detection due to its programmability, high fidelity, and signal amplification. Herein, a novel universal CRISPR/Cas12a-G4 DNAzyme-TMB (Cas-GT) enzymatic biosensing platform was constructed. This platform regulates the catalytic function of G4 DNAzyme through the trans-cleavage activity of Cas12a, achieving an "off/on" response of the TMB-H2O2 enzymatic signal for nucleic acid, protein, and metal ion targets. In proof-of-concept experiments, the detection limit of Cas-GT for in vitro transcribed SARS-CoV-2 RNA reached as low as 100 aM and it distinguished clinical positive from negative patients with good diagnostic performance (AUC = 0.9420). It is also suitable for protein targets, enabling quantitative analysis of prostate-specific antigen (PSA) within the range of 0-100 ng mL-1, with results highly consistent with clinical chemiluminescence immunoassay (CLIA). For small-molecule targets, Cas-GT exhibited good universality, achieving quantitative detection of Hg2+ within the range of 0.06-4 ng mL-1, with recovery rates of 93%-107.4% in spiked river water samples, showing no significant difference from ICP-MS (P = 0.9968). In summary, Cas-GT is a simple, sensitive, rapid, and label-free enzymatic biosensing platform with significant potential for clinical biomarker detection and environmental pollution monitoring.

CRISPR/Cas12a-based tag-free fluorescent biosensor using G-quadruplex specific thioflavin T for detection of oncogenic microRNAs.

MicroRNAs (miRs) are central regulators of tumor initiation and progression, and their aberrant expression patterns have been identified as clinically valuable biomarkers for the early diagnosis of malignancies and prognostic evaluation. Here, we report tag-free fluorescence biosensing platform for the detection of circulating miRs in serum, targeting microRNA-21 (miR-21) and microRNA-10b (miR-10b) as clinically relevant oncogenic markers. The assay integrates CRISPR/Cas12a-mediated signal regulation with complementary strand (CS)-mediated target recognition. In this strategy, target miRs hybridize with the CS, thereby preventing CS-mediated activation of the Cas12a-crRNA complex. As a result, Cas12a collateral cleavage is suppressed, the G-quadruplex reporter remains intact, and Thioflavin T fluorescence is enhanced. The platform demonstrated excellent sequence discrimination capability, effectively distinguishing closely related and mismatched targets. Sensitive quantification was achieved with limits of detection of 1.4 nM for miR-21 and 852 pM for miR-10b. Importantly, robust analytical performance was maintained in complex biological matrices, confirming its applicability to serum samples. Collectively, this CRISPR/Cas12a-enabled fluorescent biosensor provides a simple approach for circulating miR detection.