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. Graphical Abstract

A CRISPR-Cas12a-Assisted Fluorescence Platform for Rapid and Accurate Detection of Nocardia cyriacigeorgica

“…We used non-canonical C-containing sequences as PAM sites to guide the Cas12a/crRNA complex to bind to the target sequence. According to previous studies, LbCas12a possessed extensive PAM sites and non-canonical Ccontaining sequences could be suboptimal PAM sites (Tran et al, 2021;Dronina et al, 2022). The application of Cas12a to nucleic acid detection relies on trans-cleavage activity.…”

Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications

“…For instance, a single crRNA processed by Cas12 RNase domain guides Cas12 with a T-rich PAM sequence to cleave dsDNA targets, generating sticky ends[ 21 ] (Figure 1 ). Cas12a cleaves both the target and non-target strands of a targeted dsDNA by a single active site in the RuvC catalytic pocket[ 22 ]. Besides Cas12, Cas14 is the smallest RNA-guided nuclease discovered to date with 400-700 amino acids and does not require a target sequence such as PAM in the ssDNA substrate[ 23 ].…”

Electrochemical Determination of Interaction between SARS-CoV-2 Spike Protein and Specific Antibodies

“…The primary principle underlying biosensing devices is the conversion of biotarget detection into an analytical signal for further analysis. A variety of molecules including enzymes [ 1 , 2 ], proteins [ 3 , 4 ], antibodies [ 5 , 6 ], and nucleic acids [ 7 , 8 ] can be used as target biomolecules, with electrochemical [ 5 , 9 , 10 ], optical [ 11 ], piezoelectric [ 12 ], surface plasmon resonance [ 13 ], and other methods being commonly used for the analytical signal registration.…”