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The current SARS-CoV-2 pandemic forms a major global health burden. Although protective vaccines are available, concerns remain as new virus variants continue to appear. CRISPR-based gene-editing approaches offer an attractive therapeutic strategy as the CRISPR-RNA (crRNA) can be adjusted rapidly to accommodate a new viral genome sequence. This study aimed at using the RNA-targeting CRISPR-Cas13d system to attack highly conserved sequences in the viral RNA genome, thereby preparing for future zoonotic outbreaks of other coronaviruses. We designed 29 crRNAs targeting highly conserved sequences along the complete SARS-CoV-2 genome. Several crRNAs demonstrated efficient silencing of a reporter with the matching viral target sequence and efficient inhibition of a SARS-CoV-2 replicon. The crRNAs that suppress SARS-CoV-2 were also able to suppress SARS-CoV, thus demonstrating the breadth of this antiviral strategy. Strikingly, we observed that only crRNAs directed against the plus-genomic RNA demonstrated antiviral activity in the replicon assay, in contrast to those that bind the minus-genomic RNA, the replication intermediate. These results point to a major difference in the vulnerability and biology of the +RNA versus −RNA strands of the SARS-CoV-2 genome and provide important insights for the design of RNA-targeting antivirals. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) infections and HIV-induced acquired immunodeficiency syndrome (AIDS) continue to represent a global health burden. There is currently no effective vaccine, nor any cure, for HIV infections; existing antiretroviral therapy can suppress viral replication, but only as long as antiviral drugs are taken. HIV infects cells of the host immune system, and it can establish a long-lived viral reservoir, which can be targeted and edited through gene therapy. Gene editing platforms based on the clustered regularly interspaced palindromic repeat-Cas system (CRISPR-Cas) have been recognized as promising tools in the development of gene therapies for HIV infections. In this review, we evaluate the current landscape of CRISPR-Cas-based therapies against HIV, with an emphasis on the infection biology of the virus as well as the activity of host restriction factors. We discuss the potential of a combined CRISPR-Cas approach that targets host and viral genes to activate antiviral host factors and inhibit viral replication simultaneously. Lastly, we focus on the challenges and potential solutions of CRISPR-Cas gene editing approaches in achieving an HIV cure. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA guide molecule, mediates a sequence-specific attack on RNA, and can be developed as an anti-coronavirus strategy. We analyzed the SARS-CoV-2 genome to localize the hypothetically best crRNA-annealing sites of 23 nucleotides based on our extensive expertise with sequence-specific antiviral strategies. We considered target sites of which the sequence is well-conserved among SARS-CoV-2 isolates. As we should prepare for a potential future outbreak of related viruses, we screened for targets that are conserved between SARS-CoV-2 and SARS-CoV. To further broaden the search, we screened for targets that are conserved between SARS-CoV-2 and the more distantly related MERS-CoV, as well as the four other human coronaviruses (OC43, 229E, NL63, HKU1). Finally, we performed a search for pan-corona target sequences that are conserved among all these coronaviruses, including the new Omicron variant, that are able to replicate in humans. This survey may contribute to the design of effective, safe, and escape-proof antiviral strategies to prepare for future pandemics. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA (dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs were designed to target highly conserved viral sequences, their efficacy may be challenged by high genetic variation in the HIV-1 genome. We therefore evaluated the breadth of these dual-gRNA combinations against distinct HIV-1 isolates, including several subtypes. Replication of nearly all virus isolates could be prevented by at least one gRNA combination, which caused inactivation of the proviral genomes and the gradual loss of replication-competent virus over time. The dual-gRNA efficacy was not affected by most single nucleotide (nt) mismatches between gRNA and the viral target. However, 1-nt mismatches at the Cas9 cleavage site and two mismatches anywhere in the viral target sequence significantly reduced the inhibitory effect. Accordingly, sequence analysis of viruses upon breakthrough replication revealed the acquisition of escape mutations in perfectly matching and most 1-nt mismatching targets, but not in targets with a mismatch at the Cas9 cleavage site or with two mismatches. These results demonstrate that combinatorial CRISPR-Cas9 treatment can cure T cells infected by distinct HIV-1 isolates, but even minor sequence variation in conserved viral target sites can affect the efficacy of this strategy. Successful cure attempts against isolates with divergent target sequences may therefore require adaptation of the gRNAs. [ABSTRACT FROM AUTHOR]