CRISPR-Cas13 as a treatment for COVID-19: is it worth the effort?

Although titanic efforts are being made for the development of treatments against the novel SARS-CoV-2 disease (COVID-19) and the approval of the recently developed vaccines by the Food and Drug Administration (FDA) and counterpart regulatory agencies such as the European Medicines Agency (EMA), in the present there are no specific treatment to fight against the virus. Therefore, the best solution for controlling the pandemic, in addition to following preventive methods, could be the development of effective therapies that allow us to stop the course of the infection, alleviating the symptoms and the sequels after viral infection.

The development of antiviral drugs is a challenging process, and some researchers suggest the use of the CRISPR-Cas system to target and degrade the SARS-CoV-2 genome. In contrast to traditional vaccines and therapies, which rely on priming the human immune system to identify viral proteins and components, therefore reducing viral entrance into the cells [1], CRISPR based therapeutics has focused on identifying and degrading the intracellular viral genome and its resulting viral mRNAs. In this regard, the identification of SARS-CoV-2 molecular characteristic is a key point for the development CRISPR-Cas targets. 

Viral genome of SARS-CoV-2 is 88% alike to bat-SL-CoVZC45 and bat-SL-CoVZXC21, and 96% identical to bat CoV RaTG13 [2], although the ~4% difference of SARS-CoV-2 from RaTG13 accounts for almost 20-50 years of evolution, being molecularly too far away to be a direct ancestor of SARS-CoV-2. Genomic-encoded proteins of SARS-CoV-2 are 79,5% and 51% related to SARS-CoV-1 and MERS-CoV, respectively [3, 4], and it uses the angiotensin converting enzyme 2 (ACE2) receptor for cellular entry [2]. CoV spike (S) protein constitutes the receptor-binding domain (RBD), interacting with the ACE2 receptor and directing the membrane fusion between the virus and the plasma membrane [5].

After admittance, the viral RNA genome is liberated into the cytoplasm and two-thirds of it is translated into two polyproteins (pp) named pp1a and pp1b, which codifies 16 non-structural proteins (NSPs) [6]. SARS-CoV-2 has particular viral replicase genes in a variable number open reading frames (ORFs), which encodes viral proteins for viral replication, nucleocapsid and spikes formation. The rest of the ORFs encode ~8 accessory proteins that intervene wit the host innate immune response [7]. After protein translation, the nucleocapsid is created by combining genomic RNA and the nucleocapsid proteins. At the end, vesicles containing the viral particles are fused to the plasma membrane to release the virus from the infected cell. 

Genomic organization of SARS-CoV-2. Alanagreh L, et al. The Human Coronavirus Disease COVID 19: Its Origin, Characteristics, and Insights into Potential Drugs and Its Mechanisms. Pathogens. 2020 Apr 29;9(5):331. doi: 10.3390/pathogens9050331.

A few months ago, Abbott et al. published a paper in Cell [8] expanding the application of CRISPR/Cas13, in addition to its diagnostic function, for therapeutic goals by their Prophylactic Antiviral CRISPR in the huMAN cells (PAC-MAN) approach. They identified two highly conserved regions in the SARS-CoV-2 genome which can be appropriate to be targeted by PAC-MAN as a potential pan-CoV inhibitory strategy: the RNA-dependent RNA polymerase (RdRp) gene that maintains the proliferation of all CoVs, and the nucleocapsid (N) gene, which encodes the capsid protein for viral packaging. In a work published by Nguyen et al., 10,333 guide RNAs (gRNAs) were designed to specifically target ten peptide-coding regions of the SARS-CoV-2 genome [9].  

Can be the CRISPR-Cas13 system efficiently applied as a treatment for COVID-19?

The Class 2 type VI CRISPR-associated RNA-guided ribonuclease Cas13 can be divided into four protein families: Cas13a (C2c2), Cas13b, Cas13c and Cas13d (CasRx). Cas13 differs from other Cas proteins because it has two higher prokaryotes and eukaryotes nucleotide binding (HEPN) domains which together form the ribonuclease-active site that allows the protein to act as an RNA-guided RNA-targeting CRISPR effector [10]. As Cas13 has the ability to efficiently target and cleave RNA in several model systems (including mammal cells), researchers suggest that it could cleave the specific sequence of SARS-CoV-2 RNA and inactivate the virus at the posttranscriptional level in mammalian cells. CRISPR-Cas13 system has already been used as a suitable tool for SARS-CoV-2 nucleic acid detection, and the FDA approved SHERLOCK as the first CRISPR-based detection kit, providing results in ~1 hour. 

Regarding CRISPR-Cas13 therapeutic application, several SARS-CoV-2 proteins have been proposed as a target for the Cas13d ribonuclease, such as the ORF1a/b which encodes the polyproteins pp1a and pp1b, the viral main proteinases 3CLpro or Mpro, and NSP5, as the elimination of all of them can inhibit the activation of viral polyproteins. Another strategy relies on the use of a death Cas13 (dCas13), a nuclease which is capable of binding to the RNA without cleaving it. Binding of dCas13 to SARS-CoV-2 RNA may interfere with its expression, preventing the transmission to remaining healthy cells in early stages and inhibiting the development of COVID-19 to severe stages. Some researchers suggest that CRISPR-Cas13d could be a beneficial, flexible and rapid novel method for treating and preventing RNA virus infections, specially COVID-19, a strategy complementary with the development of suitable preventive and therapeutic approaches comprising vaccines, monoclonal antibodies, peptides, interferon therapies, and small molecule drugs to overcome SARS-CoV-2.

Alternatives for CRISPR-Cas13-mediated targeting of SARS-CoV-2 genome by using PAC-MAN. Abbott, T. R., et al. Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. (2020) Cell, 181(4), 865–876.e12. https://doi.org/10.1016/j.cell.2020.04.020

Nevertheless, we should keep in mind that there are huge limitations when adapting the CRISPR-Cas technology to the clinic. The most important is related to the in vivo delivery efficiency. Although there are several methods for transferring the CRISPR systems into the target cells like lipofection, electroporation, nucleofection or microinjection, viral vector constitutes the best approach for the delivery of CRISPR components into live tissue. In their PAC-MAN approach, Abbott et al. used the Cas13d variant due to its small size; it can be delivered into mammalian cells as an ‘all-in-one’ adeno-associated virus (AAV). Up to three gRNAs targeting different peptide-coding regions of the SARS-CoV-2 genome can be packaged into one AAV. Moreover, there are several AAV serotypes highly specific to the lungs than can be employed for targeted transmission of the CRISPR-Cas13d system [9]. 

Furthermore, the collateral cleavage activity of the Cas13, although having minimal off-target effects on the host transcriptome in mammalian cells [11, 12], may lead to unwanted and unpredicted mutations, so its different side effects should be studied. 

Due to those limitations and despite its enormous potential clinical applications, CRISPR-Cas13 has received less attention as a therapeutic option against SARS-CoV-2. More efforts are needed to develop better delivery strategies, to assure the lack of toxicity within the mammalian cells and to get the approval by the appropriate regulatory agencies. 

References:

1. Rappuoli, R., Glycoconjugate vaccines: Principles and mechanisms. Sci Transl Med, 2018. 10(456).

2. Zhou, P., et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 2020. 579(7798): p. 270-273.

3. Banerjee, A., et al., Bats and Coronaviruses. Viruses, 2019. 11(1).

4. Guo, Y.R., et al., The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak - an update on the status. Mil Med Res, 2020. 7(1): p. 11.

5. Simmons, G., et al., Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry. Proc Natl Acad Sci U S A, 2004. 101(12): p. 4240-5.

6. Zumla, A., et al., Coronaviruses - drug discovery and therapeutic options. Nat Rev Drug Discov, 2016. 15(5): p. 327-47.

7. Wu, A., et al., Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China. Cell Host Microbe, 2020. 27(3): p. 325-328.

8. Abbott, T.R., et al., Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. Cell, 2020. 181(4): p. 865-876 e12.

9. Nguyen, T.M., Y. Zhang, and P.P. Pandolfi, Virus against virus: a potential treatment for 2019-nCov (SARS-CoV-2) and other RNA viruses. Cell Res, 2020. 30(3): p. 189-190.

10. Shmakov, S., et al., Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell, 2015. 60(3): p. 385-97.

11. Abudayyeh, O.O., et al., RNA targeting with CRISPR-Cas13. Nature, 2017. 550(7675): p. 280-284.

12. Konermann, S., et al., Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell, 2018. 173(3): p. 665-676 e14.

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