A short overview of CRISPR-Cas technology and its application in viral disease control

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) together with CRISPR-associated (Cas) proteins have catalysed a revolution in genetic engineering. Native CRISPR-Cas systems exist in many bacteria and archaea where they provide an adaptive immune response through sequence-specific degradation of an invading pathogen's genome. This system has been reconfigured for use in genome editing, drug development, gene expression regulation, diagnostics, the prevention and treatment of cancers, and the treatment of genetic and infectious diseases. In recent years, CRISPR-Cas systems have been used in the diagnosis and control of viral diseases, for example, CRISPR-Cas12/13 coupled with new amplification techniques to improve the specificity of sequence-specific fluorescent probe detection. Importantly, CRISPR applications are both sensitive and specific and usually only require commonly available lab equipment. Unlike the canonical Cas9 which is guided to double-stranded DNA sites of interest, Cas13 systems target RNA sequences and thus can be employed in strategies directed against RNA viruses or for transcriptional silencing. Many challenges remain for these approach, including issues with specificity and the requirement for better mammalian delivery systems. In this review, we summarize the applications of CRISPR-Cas systems in controlling mammalian viral infections. Following necessary improvements, it is expected that CRISPR-Cas systems will be used effectively for such applications in the future.

Keywords: CRISPR; Cas13 protein; Detection kit; Viral RNA; Viral disease.

Fig. 1

Bacterial CRISPR-Cas9 systems behave as an adaptive immune response against invading bacteriophages. Following infection (Phase 1), Cas1 and Cas2 mediate incorporation of short sequences of the viral genome as spacers within the bacterial CRISPR locus. At re-exposure (Phase 2), the CRISPR locus gets expressed as pre-crRNA, along with tracrRNA. The pre-crRNA is processed to yield guide RNAs (gRNAs) which bind the ribonucleoprotein Cas9 and target this complex to complementary sequences of the infiltrating bacteriophage genome, prompting its Cas9-mediated cleavage. Reprinted from “CRISPR-Cas9 Adaptive Immune System of Streptococcus pyogenes Against Bacteriophages”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates

Fig. 2

Schematic of viral nucleic acid degradation using Cas9 and Cas13. Created with BioRender.com

Fig. 3

Schematic of viral nucleic acid detection with CRISPR Cas12 and 13. Nucleic acid targets can be amplified by LAMP/RPA (DNA) and RT-LAMP/RPA (RNA). Cas12- and Cas13-crRNA complexes cleave introduced target-specific fluorescent probes (F) on DNA and RNA, respectively, removing quencher (Q) moieties and producing detectable fluorescent signals. Created with BioRender.com