Psmcas13b as a Tool for RNA-Based Therapeutics and Disease Treatment

Based on the free article:
https://www.mdpi.com/2306-5354/9/7/291

In recent decades, RNA viruses have emerged as a major threat to public health, causing global health crises. Despite significant advancements in diagnostic methods and antiviral therapies for combating these viruses, traditional detection methods such as ELISA and RT-qPCR are often time-consuming and limited in their sensitivity. While vaccines and drugs have proven effective in preventing and treating viral infections, their research and development cycles can be lengthy. However, in recent years, the CRISPR-Cas13 system has emerged as a promising tool for RNA virus diagnosis and therapy. This system has accurate RNA-targeting capabilities, making it stand out from other CRISPR-Cas systems. This review explores the potential of the CRISPR-Cas13 system for combating RNA viruses, including some of the most medically significant ones like SARS-CoV-2, dengue virus, and HIV-1, with a focus on its use in both diagnostics and therapeutics.

Throughout history, humans have suffered from numerous infectious diseases caused by RNA viruses. The recent COVID-19 outbreak serves as a stark reminder of this fact, having claimed over six million lives and infected nearly 530 million people as of 31 May 2022. Other RNA viruses, such as the dengue virus, which infects 96 million people across more than 100 countries annually, and the human immunodeficiency virus (HIV), which has infected over 37 million people worldwide, also pose significant threats to global public health and economic stability. The high evolutionary rates of RNA viruses make it challenging to detect their infections, leading to delayed treatment and worse outcomes. As a result, there is an urgent need for effective diagnostic and therapeutic methods to combat RNA viruses.

The Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR-Cas) systems are known as bacterial immune systems that provide adaptive immunity to hosts against invading nucleic acids, including viruses. While the DNA-targeting CRISPR-Cas9 system has proven useful, the recently discovered RNA-targeting CRISPR-Cas13 system has shown great potential in gene silencing without affecting the entire genome. In vivo studies have demonstrated that the CRISPR-Cas13 system is more effective in knocking down RNA than traditional RNAi technology. Additionally, in vitro studies have revealed that the CRISPR-Cas13 system has collateral activity that can cleave RNA promiscuously. Researchers have harnessed the unique characteristics of the CRISPR-Cas13 system to combat RNA viruses such as SARS-CoV-2, dengue virus, and HIV-1. In this article, we provide a perspective on the use of the CRISPR-Cas13 system for the diagnosis and treatment of RNA viruses, highlighting its potential in both diagnostics and therapeutics.

The CRISPR-Cas13 system is a Type VI Class 2 CRISPR-Cas system consisting of two main components, a single Cas13 nuclease and a corresponding CRISPR RNA (crRNA). Unlike Class 1 CRISPR-Cas systems, which are made up of multiple subunits, Cas13 proteins are classified into specific types, including Cas13a, Cas13b (Cas13b1, Cas13b2), Cas13c, Cas13d, Cas13x, and Cas13y, according to a phylogenetic study. Cas13 proteins contain two essential higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that are crucial for RNA degradation. Mutations in HEPN domains can result in a catalytically inactive protein that retains its ability to bind target RNA, a feature that is useful in RNA imaging, tracking, and modification. The CRISPR-Cas13 system can nonspecifically degrade single-strand RNA (ssRNA) in vitro when activated by target RNA, similar to the CRISPR-Cas12 system that has single-strand DNA collateral cleavage activity. This method is commonly used in diagnostics. However, in vivo, the CRISPR-Cas13 system cleaves target RNA strands in a sequence-specific manner without promiscuous cleavage ability, making it a valuable tool in disease and antiviral therapies.

Cas13a, also known as C2C2, was discovered by Zhang’s group in 2015 through a computational pipeline strategy. It was later identified as a single-component programmable RNA-guided, RNA-targeting CRISPR effector. To be programmed to degrade target ssRNA, the CRISPR/LshCas13a system requires the specification of a 28 nt sequence on the crRNA and a 3′ non-G nucleotide protospacer-flanking sequence (PFS) motif adjacent to the protospacer target. However, a lateral study found that the CRISPR/LwaCas13a system does not require a PFS motif, which enhances the flexibility of Cas13a. In 2017, Zhang et al. introduced Cas13b, including two variants, Cas13b1 and Cas13b2, corresponding to the related accessory proteins Csx27 and Csx28, through a computational-sequence database-mining approach. It is noteworthy that Csx27 represses Cas13b activity, while Csx28 enhances it. Unlike Cas13a, Cas13b is a larger protein with 1150 amino acids and requires a double-sided PFS, 5′ PFS of D (A, U, or G), and 3′ PFS of NAN or NNA to maximize its targeting ability.

Detection

Diagnostic tests for RNA-based viruses are classified into two main types: immunological and molecular methods. ELISA is an immunological method that can directly detect virus antigens or antibodies in infected people’s blood. It’s been used to detect SARS-CoV-2 spike protein, dengue NS1 protein, and HIV p24 antigen. The molecular assays, based on RT-qPCR technology, detect RNA, and are the gold standard for SARS-CoV-2 detection. Although ELISA and RT-qPCR are widely used, they’re time-consuming and require trained personnel. To meet the demand for higher sensitivity and specificity, researchers have explored novel detection methods, including CRISPR-Cas systems. While CRISPR-Cas systems are gene-editing tools, CRISPR-Cas13 is especially useful for RNA virus detection because of its highly accurate RNA-targeting ability. The CRISPR-Cas13 system can identify RNA samples in a point-of-care (POC) testing format, without the need for expensive equipment or skilled personnel. It’s rapid, highly sensitive, selective, and cost-effective, making it ideal for detecting various RNA samples.

In 2017, Zhang’s team developed a sensitive, quick, and inexpensive nucleic acid detection method called Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), which uses CRISPR-Cas13a. When CRISPR-Cas13a is combined with a properly designed target RNA recognition, it can use its promiscuous ribonuclease activity with a fluorescence reporter RNA. Single-molecule sensitivity has been achieved by the combinational amplification method of reverse transcriptase and recombinase polymerase amplification (RT-RPA) and T7 RNA polymerase transcription combined with the signal report of the collateral RNA cleavage ability of CRISPR-Cas13a. They conducted clinical validation of a two-step assay for detecting SARS-CoV-2 RNA based on this method. The S gene provided the best sensitivity performance for both lateral-flow strips and fluorescence tubes based on a comparison analysis of the limit of detection (LoD) of four selected gene regions of the SARS-CoV-2 genome map. 154 clinical samples showed 88% sensitivity on lateral-flow strips and 96% sensitivity on fluorescence signals with a detection limit of 42 RNA copies per reaction. SHERLOCK also detected other common human coronaviruses with no cross-reactivity, indicating high specificity. Furthermore, SHERLOCK was able to produce a reliable detection result for strongly positive samples in as little as 35 minutes, which is significantly shorter than RT-qPCR (excluding RNA extraction time) by more than 120 minutes.

Zhang’s group developed a method to detect dengue viruses using the CRISPR-Cas13 system after successfully creating a detection method for RNA viruses using SHERLOCK. They used multiple crRNAs with intentionally designed mismatches in spacer sequences to increase sensitivity and specificity, and SHERLOCK was able to detect corresponding dengue virus strains with high signal. The researchers also developed SHERLOCKv2 by combining Cas13a and Cas13b with other CRISPR-associated protein nucleases such as Cas12 and Csm6 to detect multiple pathogens, including dengue virus, with a quantitative measurement of as low as 2aM. The four Cas proteins used in the experiment had distinct RNA dinucleotide cleavage preferences with reporters AU, UC, AC, and GA, which made them orthogonal in the same test tube. The combination of Cas13 and Csm6 resulted in a 3.5-fold increase in signal sensitivity.

Therapeutics

Current strategies for combatting RNA viruses involve vaccination and small molecule antiviral drugs. While vaccination has been effective in preventing viral infections, it has only been successful for a limited number of viruses due to the high evolutionary rates of RNA viruses that often lead to vaccine failure. Antiviral drugs, on the other hand, require a thorough understanding of the virus and its mechanisms, making it a time-consuming process. Additionally, drug resistance and pathogen mutations remain a concern. Therefore, there is an urgent need for adaptable antiviral therapeutic platforms to combat RNA viruses. While CRISPR-Cas9 and CRISPR-Cas12 can only target certain RNA virus classes, the CRISPR-Cas13 system can target any RNA virus. Its specific RNA targeting ability makes it a potential candidate for a “virus against virus” strategy.

In 2020, Abbott and his team developed a method called PAC-MAN, which used CRISPR-Cas13d to genetically prevent SARS-CoV-2 infection. They identified highly conserved regions in the virus’s genome and designed a pool of six crRNAs that were able to effectively target and cleave these sequences, resulting in a 91% targeting efficiency. The researchers found that the RdRP and N genes of SARS-CoV-2 were perfect targets for the CRISPR-Cas13d system, as they were essential for viral replication and packaging. Using this method, the team was able to achieve a 90% reduction in SARS-CoV-2 mRNA expression in lung epithelial cells. Targeting and degrading the virus’s RNA using CRISPR-Cas13d showed great potential in inhibiting SARS-CoV-2.

In another study, researchers used the CRISPR-Cas13 system to target the dengue virus, which currently has no specific therapy. Li et al. found that cleaving the dengue virus NS3 gene with CRISPR-Cas13a could effectively inhibit viral replication. They identified 10 vulnerable sites for targeting using the CRISPR-Cas13a system, and the designed crRNAs were formulated with the LwaCas13a protein to transfect Vero cells infected with DENV-2. After testing various crRNAs, they found that NS3-crRNA was the most effective, leading to a 95% decrease in viral RNA copy after three days post-transfection of the NS3-crRNA-Cas13a complex. Another study by Singsuksawat et al. used Cas13b RNP with a short spacer to enhance knockdown activity. They identified 8681 crRNA as the most efficient specific to DENV2-16681. They re-engineered a retrovirus to deliver Cas13b RNP to primary human target cells, achieving strong suppression of dengue infection even with small amounts of PspCas13b.

Yin et al. utilized the CRISPR-Cas13 system to target various regions of the HIV-1 virus, resulting in significant reduction of viral RNA, YFP fluorescence, and HIV-1 gag expression. Furthermore, the CRISPR-Cas13a system efficiently targeted and destroyed RNA from transfected plasmid DNA and integrated viral DNA. The researchers concluded that this system holds great potential as a novel tool to combat HIV-1. Similarly, Kulkarni’s group employed the Cas13d protein with multiple crRNAs targeting HIV-1 gag, pol, and cPPT genes. The combination of Cas13d and multiple crRNAs achieved over 90% inhibition efficiency in HIV replication. Interestingly, they found that Cas13d was more effective in the nucleus than in the cytoplasm, suggesting that it could be a more powerful treatment tool due to its better RNA-silencing performance in the nucleus compared to RNAi.

Conclusion

RNA viruses have caused contagious diseases for years, with the SARS-CoV-2, dengue virus, and HIV-1 being the most prevalent. The CRISPR/Cas13 system has shown its potential in RNA virus diagnostics and therapeutics. The CRISPR/Cas13 system can recognize and cleave ssRNA promiscuously, allowing for several nucleic acid detection platforms to be developed. To improve the sensitivity of CRISPR/Cas13-based detection, amplification techniques have been incorporated into the detection system, including PCR, RPA, LAMP, transcription amplification, and others. Amplification-free detection platforms have also been explored to prevent time waste and amplification errors. Rapid virus evolution and mutation have led to the development of serological antigen tests as a complement to improve diagnostic accuracy. Different signal output modes, such as lateral flow strips and mobile phone cameras, can be used as fluorescence readers for the CRISPR/Cas13-based nucleic acid detection platforms, making them beneficial in point-of-care pathogen testing.

The CRISPR-Cas13 system has shown potential in combating RNA viruses through the design of crRNAs that correspond to conserved genes of a specific virus. Several Cas13 proteins have been used to effectively target different RNA viruses including SARS-CoV-2, dengue virus, and HIV. However, it is important to note that the delivery methods and safety concerns of the CRISPR-Cas13 system in vivo are still underdeveloped. Nevertheless, the CRISPR-Cas13 system is a promising tool for the detection and treatment of RNA viruses and has the potential to exhibit distinct characteristics in this new age. Additionally, the collateral activity of C2C2, a Cas13a variant, has been observed in vitro and may serve as a reliable gene-editing therapeutic tool. Overall, there is room for improvement in the CRISPR-Cas13 system’s further applications, but it presents a feasible option for both detection and treatment of RNA viruses.

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