Advanced CRISPR-Cas Effector Enzyme-Based Diagnostics for Infectious Diseases, Including COVID-19

Abstract

Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics.

Exploiting the Specificity of CRISPR/Cas System for Nucleic Acids Amplification-Free Disease Diagnostics in the Point-of-Care

“…Cas12a is simpler to employ, unlike Cas12b, which requires the presence of tracrRNA to achieve interference. 37 Cas12b has not yet been extensively studied in diagnostics due to the requirement for higher temperatures (at 48°C); however, efforts have been made to engineer the enzyme for operation at lower temperatures. 38 The high efficiency trans-cleavage activity (around 1250 rotations per second) and crRNAdependent sequence specificity exhibited by Cas12a have been employed to detect nucleic acids with excellent specificity, sensitivity, and speed.…”

“…Cas12a and Cas12b are among the most common subtypes. Cas12a is simpler to employ, unlike Cas12b, which requires the presence of tracrRNA to achieve interference . Cas12b has not yet been extensively studied in diagnostics due to the requirement for higher temperatures (at 48°C); however, efforts have been made to engineer the enzyme for operation at lower temperatures .…”

Research progress of CRISPR/Cas systems in nucleic acid detection of infectious diseases

“…In addition to recognizing target dsDNA, Cas12a recognizes target ssDNA and specifically cleaves near its 22 nd position; this process does not require PAM [38] (Figure 1c). Cas12f, formerly known as Cas14, is similarly able to recognize and specifically cleave target ssDNA, again without the need of PAM [39]. Cas12f is relatively short, with approximately 400–700 amino acids, which is only half that of other Cas12 proteins and one‐third that of Cas9.…”

Immunopathogenesis of Orthopoxviridae: Insights into Immunology from Smallpox to Monkeypox (Mpox)

“…More recently, clustered regularly interspaced short palindromic repeats (CRISPR), and other biosensors (e.g., CRISPR/Cas12b) are in development that could potentially be suitable for viral detection. These could be more cost-effective than traditional diagnostics and potentially more accurate than lateral flow device (LFD) testing [59][60][61][62][63].…”