Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review)

Abstract

Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.

Keywords: Bioanalytical system; COVID-19; CRISPR-Cas; CRISPR–Cas12a; DNA-biosensors; SARS-CoV-2 virus.

Fig. 1

Performance of basic CRISPR-Cas system: a Overview of schematic CRISPR definition; b Cas proteins are nucleases, endoribonucleases, helicases, or/and integrases: (1) single-strand DNA (break) by nuclease activity; (2) double-strand DNA (break) by nuclease activity; (3) double-strand DNA unwinding by helical activity; c Structure of Cas protein, guide RNA (crRNA) and target DNA complex; d Performance of four-part CRISPR-Cas action mechanism in the prokaryotic cell: 1. Stage—adaptation; 2–3. Stages—expression; 4. Stage—interference

Fig. 2

CRISPR-Cas12a resembles the beak structure: the active center is suppressed in the closed position, and the active center is released—in the open position. The a N-terminal recognition (REC) region is divided into two [Rec1 (13 α helices) and Rec2 (10 α helices and 2 β strands)] alpha-helical domains that form an antiparallel sheet at the top of the structure. The C-terminal NUC lobe is divided into Wedge [WED (7 α helices and 2 β strands)], PAM-interacting [PI (7 α helices and β hairpin)] and an endonuclease domain involved in DNA repair RuvC (three motifs (RuvC I–III), which form active endonuclease center) and Nuc at the bottom of the structure. Modified bridge helix (BH consist of Arg951 and Arg955 which interact with the phosphate backbone of the target DNA strand) region is in the middle of NUC and REC lobes. The cleavage mechanism: a cis-cleavage in RuvC domain; b cis- and trans-cleavages in RuvC domain; c E-CRISPR biosensor prototype based on CRISPR-Cas12a system: C1—target dsDNA detection by cis- cleavage when target dsDNA is immobilized on AuNP as analyte. C2—target dsDNA detection by trans-cleavage when ssDNA is immobilized on AuNP and dsDNA is analyte. C3—target dsDNA detection by trans-cleavage when CRISPR-Cas12a is immobilized on AuNP and dsDNA is analyte. C4—dsDNA detection by trans-cleavage and additional effector when ssDNA is immobilized on AuNP and dsDNA as analyte. The CRISPR-Cas12a system can be fused with some newly designed enzymes like polymerases, other nucleases, or fluorescent proteins as additional effectors. Afterward, modified Cas protein in the CRISPR-Cas system can be used to transport those effectors to a specific DNA sequence for transcription, specific hydrolysis, visualization, or another practical purpose target. Together, through the examples detailed above, we have illustrated integrating CRISPR-Cas systems into different types in vivo biological sensing scenarios as well as emerging monitoring points compassionate and selective diagnostic programs determination of nucleic acids, proteins, and other small molecules