A CRISPR Test for Detection of Circulating Nuclei Acids

Abstract

Emerging CRISPR-based nucleic acid detection shows great promise in molecular diagnosis of diseases. CRISPR-Cas12a can sensitively and specifically detect human papillomavirus (HPV) DNA in anal swabs. However, the current CRISPR-Cas12a system needs auxiliary and expensive equipment, which limit its application as a point-of-care (POC) diagnostic tool. This study aimed to develop CRISPR-Cas12a as a POC test to directly target plasma for circulating HPV DNA detection by immediately reading results with naked eyes. Cell-cultured supernatants of either HPV16- or 18-positive cancer cells were treated with lysis buffer followed by isothermal amplification without DNA isolation. Cas12a, crRNA, and fluorescent-biotin reporters were incubated with the lysates. Our data showed that integrating CRISPR-Cas12a with lateral-flow strips could directly and specifically detect HPV16 and 18 in the liquid samples with the same limit of detection (0.24 fM) as did polymerase chain reaction but requiring less time. Furthermore, the CRISPR-Cas12a system could rapidly detect presence of HPV16 and HPV18 in plasma samples of 13 of 14 and 3 of 10 the patients with histopathological diagnosis of cervical cancer, respectively. Therefore, a CRISPR-Cas12a–based POC system was developed for conveniently detecting circulating nuclei acid targets in body fluids without requiring technical expertise and ancillary machineries.

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…In most of these platforms, a commercially available universal test strip, the HybriDetect-Universal Lateral Flow Assay Kit, was used. This dipstick was originally designed for qualitative or even quantitative rapid testing of proteins, antibodies or gene amplicons, but has been adapted to function in several LFA based CRISPR sensing methods ( Bai et al, 2019 ; Chang et al, 2019 ; Gootenberg et al, 2018 ; Kaminski et al, 2020 ; Mukama et al, 2020b ; Sullivan et al, 2019 ; Tsou et al, 2019 ). This platform offers a Streptavidine line as well as an antibody line that can capture anti-FITC coated AuNPs ( Fig.…”

Magnetic Bead-Quantum Dot (MB-Qdot) Clustered Regularly Interspaced Short Palindromic Repeat Assay for Simple Viral DNA Detection

“…Unlike PCR that requires ultra-purified buffer solutions, 44 , 45 one of the unique advantages of CRISPR-based detection is that the collateral cleavage process is not affected much by the presence of nucleases in bodily fluids. For example, numerous studies have shown the sensitive detection of nucleic acid samples in urine, 46 saliva, 17 and plasma 47 samples without stringent purification. However, to meet the requirements of “real-world” POC diagnostics, a nucleic acid concentration step may still be needed as the lysis process could result in the dilution and loss of target samples, especially when the viral load is low.…”

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

“…17,39 An advantage of the IMPACT chip compared to other detection apparatuses such as the SHERLOCK test strip is that it is a fully enclosed system without extra packaging need. 40,41 This is advantageous as the treated micropillars are sealed before molecular diagnostics. The reporter probes are immobilized within the channel, avoiding degradation issues from outside contaminants.…”