Abstract
Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most functional and widely used tools for RNA and DNA manipulation in multiple organisms. This approach has provided an unprecedented opportunity for creating simple, inexpensive, specific, targeted, accurate, and practical manipulations of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1), and vaccinia virus. Furthermore, this method can be used to make an effective and precise diagnosis of viral infections. Nevertheless, a valid and scientifically designed CRISPR system is critical to make more effective and accurate changes in viruses. In this review, we have focused on the best and the most effective ways to design sgRNA, gene knock-in(s), and gene knock-out(s) for virus-targeted manipulation. Furthermore, we have emphasized the application of CRISPR technology in virus diagnosis and in finding significant genes involved in virus-host interactions.
Highlights
Introduction iationsThe diversity of viral genome organizations and their replication systems is the most remarkable aspect of viruses’ molecular biology
A study of a CRISPR-wide SARS-CoV-2 genome screen showed that the SWI/SNF chromatin remodeling complex, the TGF-β signaling pathway, alarmin, HMGB1 and H3.3 chaperone complex are the main factors related to the pathogenicity of this virus [116]
Cas13, the ability of targeted A + I (G) conversion in RNA was achieved in Cas13b [143]; Cas13c: Recently identified, and its performance is under investigation [142]; Cas13d: It has very high efficiency and does not have the limitation of the protospacer flanking sequence (PFS) [144]
Summary
SgRNA is a single RNA with stem loop(s) and custom-designed nucleotides to facilitate attachments to the target region; Cas9-targeted endonuclease recognizes targeted sequences with sgRNA. It has been shown that if the sgRNA length is reduced to 17 nucleotides (truncated sgRNA), it does not disrupt the system performance and increases the system specificity, leading to decreased off-target effects elsewhere in the genome [45]. The cleaved site would be located within the exonic region or the splice site to have a complete loss of function [47,48] This design can be performed on functional domains of the protein, leading to an efficient inactivation of the protein [49]. The percentage of GC counts in the 20-nucleotide sequence complementary to the sgRNA target region, if more or less than 40–70%, may affect activity, efficiency, accuracy, and the off-targeting effect. Many software packages are introduced to facilitate sgRNA design by incorporating many of these factors and offering predicted functional sgRNAs (Table 2)
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