ABSTRACT
CRISPR-Cas is an adaptive immune system that prevents bacteria and archea from nucleic acids invasion such as viral genomes. The ability of the CRISPR-Cas technology to effectively and precisely cut a targeted genomic DNA region was exploited to develop powerful genome editing tools that were adapted for a wide range of applications, revolutionizing biological sciences. The CRISPR-Cas system consists of a Cas endonuclease triggered by a RNA guide for highly specific cleavage of targeted DNA or RNA sequences. In addition to the target specific cleavage, some Cas enzymes, including Cas12a and Cas13a, display a collateral trans-cleavage activity that allows the cleavage of all surrounding single-stranded nucleic acids. These biosensing activities of CRISPR-Cas systems, based on target specific binding and cleavage, are promising tools to develop accurate diagnostic methods to detect specific nucleic acids. CRISPRCas could therefore be used to diagnose a wide variety of diseases. In the current review we propose to describe the more significant advances for virus detection based on CRISPR-Cas systems.
CRISPR-Cas est décrit comme un système immunitaire adaptatif qui permet aux bactéries et aux archées de se défendre contre les agressions virales. La technologie dérivée de ces systèmes CRISPR-Cas, qui permet de cliver précisément une séquence génomique, est désormais la base de puissants outils de biologie moléculaire et d'édition des génomes. Les « ciseaux moléculaires ¼ CRISPR-Cas utilisent des endonucléases Cas, programmées et activées avec un ARN guide, pour couper spécifiquement une séquence cible ARN ou ADN. Certaines de ces enzymes Cas, notamment Cas12a et Cas13a, présentent au-delà de cette activité de coupure dirigée par un guide ARN, une activité générique de clivage collatéral en trans de toutes séquences nucléiques rencontrées. Ces différentes activités des systèmes CRISPR-Cas ont pu être exploitées pour développer des outils prometteurs de diagnostic moléculaire. Si les applications sont très nombreuses dans différents domaines, nous proposons ici d'illustrer le potentiel de ces approches basées sur CRISPR-Cas dans le cadre du diagnostic en virologie.
Subject(s)
Bacteria , CRISPR-Cas Systems , Bacteria/genetics , DNA , Endonucleases/genetics , RNAABSTRACT
Hepatitis B (HBV) infection is a major public health concern. Perinatal transmission of HBV from mother to child represents the main mode of transmission. Despite the existence of effective immunoprophylaxis, the preventive strategy is inefficient in neonates born to mothers with HBV viral loads above 2 × 105 IU/mL. To prevent mother-to-child transmission, it is important to identify highly viremic pregnant women and initiate antiviral therapy to decrease their viral load. We developed a simple innovative molecular approach avoiding the use of automatic devices to screen highly viremic pregnant women. This method includes rapid DNA extraction coupled with an isothermal recombinase polymerase amplification (RPA) combined with direct visual detection on a lateral flow assay (LFA). We applied our RPA-LFA approach to HBV DNA-positive plasma samples with various loads and genotypes. We designed a triage test by adapting the analytical sensitivity to the recommended therapeutic decision threshold of 2 × 105 IU/mL. The sensitivity and specificity were 98.6% (95% CI: 92.7-99.9%) and 88.2% (95% CI: 73.4-95.3%), respectively. This assay performed excellently, with an area under the ROC curve value of 0.99 (95% CI: 0.99-1.00, p < 0.001). This simple method will open new perspectives in the development of point-of-care testing to prevent HBV perinatal transmission.