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. 2022 Feb 14;14(2):385.
doi: 10.3390/v14020385.

In Silico Prediction and Selection of Target Sequences in the SARS-CoV-2 RNA Genome for an Antiviral Attack

Mouraya Hussein  1 , Zaria Andrade Dos Ramos  1 , Ben Berkhout  1 , Elena Herrera-Carrillo  1
Affiliations

In Silico Prediction and Selection of Target Sequences in the SARS-CoV-2 RNA Genome for an Antiviral Attack

Mouraya Hussein et al. Viruses. .

Abstract

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.

Keywords: COVID-19; CRISPR-Cas13d; SARS-CoV-2 genome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Comparison of the RNAi and CRISPR-Cas pathways. (A) shRNAs are expressed in the nucleus, and transported to the cytoplasm, where they are processed by Dicer into mature siRNA duplexes. siRNA duplexes are subsequently incorporated into the RNA-induced silencing complex (RISC), and one strand of the duplex directs this complex toward complementary mRNA targets that are inactivated by cleavage. (B) CRISPR-Cas13d activity in mammalian cells requires the expression of a CRISPR-RNA (crRNA) and the Cas endonuclease to form a complex that targets complementary RNA. Complementary RNA targets are inactivated by Cas cleavage. RNA cleavage is mediated by two nuclease domains (HEPNs; shown as two scissors).
Figure 2
Figure 2
The RNA genomes of all seven human coronaviruses. The genome size is indicated behind the virus names (in kb), and relates to the corresponding reference strains (GenBank). All genomes have a 5′UTR and 3′UTR (black box), ORF1a/b (yellow box) encoding polyprotein 1a and 1ab, spike (S) gene (green box), envelop (E) gene (purple box), membrane (M) gene (blue box), and nucleocapsid (N) gene (orange box). Hemagglutinin esterase (HE) is an additional structural gene of HCoV-HKU1 and HCoV-OC43 (pink box). HCoV-OC43 encodes additional non-structural genes (dark grey boxes): 2, 12.9, and I. Accessory genes vary from virus to virus, and are shown in the light grey boxes.
Figure 3
Figure 3
Schematic of the SARS-CoV-2 replication cycle. Upon virus binding to the ACE-2 receptor, plus-strand genomic RNA is released in the cytoplasm, and subsequently translated into the two polyproteins pp1a and pp1ab, with expression of the latter depending on a programmed −1 ribosomal frameshift at the ORF1a/ORF1b overlap region. Then, pp1a and pp1ab are proteolytically cleaved to generate 16 non-structural proteins (nsps). Several of these proteins form the replication complex that drives the synthesis of minus-strand genomic RNA, which, in turn, is copied into new plus-strand genomic RNAs that can be packaged in new virions. Discontinuous transcription generates a set of 3′ co-terminal sub-genomic mRNAs with an identical 5′UTR (shown in orange) and 3′UTR (shown in blue). The sub-genomic RNAs are translated into structural and accessory proteins that will form the new virions. Assembly and budding of the virions take place at the ER-Golgi intermediate compartment (ERGIC). Nascent virions are released from the cell via exocytosis.
Figure 4
Figure 4
Design of optimal Cas13d crRNAs against SARS-CoV-2 RNA. (A). Score distribution of crRNAs along the SARS-CoV-2 RNA genome (MN908947). crRNA guide score is assigned to each crRNA along the complete SARS-CoV-2 genome (0–30 kb). Guide scores range from 0 to 1, with higher scores being indicative for higher predicted knockdown efficacy. The crRNAs are grouped in targeting efficacy quartiles Q1–Q4, with Q4 representing the best guide RNAs (guide score closer to 1). The top crRNAs with the maximal guide score of 1 are marked in the top orange box. (B). SARS-CoV-2 RNA genome diversity. The Shannon entropy along the RNA genome varies from 0 to 1, where lower genetic diversity gives values closer to 0.
Figure 5
Figure 5
Candidate target regions in the RNA genomes of SARS-CoV-2 and all other human coronaviruses. (A) List of conserved crRNAs that target the SARS-CoV-2 genome with the best predicted knockdown efficacy (guide score ≥ 0.8). (B) Top: SARS-CoV-2 genome organization, including target regions for CRISPR-Cas13d and RNAi. Solid triangle: conserved Cas13d crRNA candidates. Open triangles: conserved RNAi candidates. RdRp: RNA-dependent RNA polymerase. &: the most conserved segment of 26-nt (15,283–15,314) among all human coronaviruses. Bottom: BLAST-mediated alignment of all other human coronavirus genomes (SARS-CoV, MERS-CoV, 229E, NL64, OC43, and HKU1), and color coding of the alignment scores. This score was computed by assigning a value to each aligned pair of bases, and counting these values over the length of the alignment.
Figure 6
Figure 6
Conserved RNA target sequences of human coronaviruses. (A) crRNAs conserved among SARS-CoV-2 and SARS-CoV. (B) Highly conserved sequence in all human coronaviruses. Red letters represent mismatches in the selected target sequence. (C) The number of conserved Cas13d and RNAi target sequences shown per pairwise alignment of all human coronaviruses (a = target length), 23-nt, and 19-nt, respectively. Hyphen (-) represents cases of non-applicability of the pairwise alignment in the indicated positions.
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