CRISPR-Cas13d effectively targets SARS-CoV-2 variants, including Delta and Omicron, and inhibits viral infection

SARS-CoV-2 has caused hundreds of millions of infections and millions of deaths globally. Rapid viral evolution has led to multiple variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and Omicron, which challenge the efficacy of vaccines, monoclonal antibodies, and antivirals due to immune escape and increased transmissibility. As an RNA virus, SARS-CoV-2 accumulates mutations quickly, necessitating adaptable antiviral strategies. CRISPR-Cas13 systems, which target RNA, offer a programmable means to directly degrade viral RNAs. The study poses the question of whether Cas13d can be designed to recognize and degrade authentic SARS-CoV-2 RNA, including VOCs such as Delta and Omicron, in infected human cells, and whether a small set of guides can provide broad coverage across variants.

Prior research established Cas13 as an RNA-guided RNase capable of targeting viral RNAs in mammalian cells and enabling antiviral applications (Cas13a/b/d). Several studies demonstrated programmable inhibition and detection of RNA viruses and early CRISPR-based strategies against SARS-CoV-2 and influenza. Compared to Cas13a/b, Cas13d is compact (967 aa), efficient, and amenable to delivery (AAV/LNP). The literature also documents the rapid emergence and epidemiology of SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, Omicron), their immune escape, and the limitations of protein-targeted interventions. However, evidence for Cas13d acting against authentic SARS-CoV-2 VOCs in infected human cells, particularly Delta and Omicron, had been lacking.

- Bioinformatic design of crRNAs: Aligned published SARS-CoV-2 genomes (NCBI and 2019-nCoVR; 1,652,991 high-quality sequences downloaded on 2021-08-01) to identify highly conserved regions, focusing on ORF1ab (NSP13 and NSP14) and nucleocapsid (N). Generated candidate 22-nt spacer sequences; excluded guides with predicted host off-target binding (allowing up to 2 mismatches) and sequences with poly-T ≥4 to avoid expression issues. - crRNA screening in vitro: Synthesized 50 crRNAs targeting conserved regions (ORF1ab and N). Employed a cell-free assay with synthetic SARS-CoV-2 RNA fragments (e.g., ORF1ab fragments F1/F2 and N) added to a 20 μl system; assessed degradation via RT-PCR/qRT-PCR to select top performers. - Plasmid construction: Inserted crRNAs into pXR004 (Addgene #109054) via BbsI cloning and verified by colony PCR/Sanger sequencing. Engineered a Cas13d expression plasmid with N-terminal His-Flag and C-terminal GFP (pADM-CMV-Cas13d-mCMV-GFP) using SgfI/MluI cloning. - Cell culture and transfection: HeLa-ACE2 and Vero-E6 cells cultured in DMEM with 10% FBS and antibiotics at 37°C/5% CO2. HeLa-ACE2 cells co-transfected with 1 μg Cas13d plasmid and 1 μg crRNA plasmid using PEI (plasmid:PEI 1:3). Transfection optimized to 70–90% efficiency by GFP and RT-PCR. - SARS-CoV-2 infection: Work performed in BSL-3. Authentic strains (SZTH-003 ancestral 614D/D614G, B.1, B.1.1.7 Alpha, B.1.351 Beta, B.1.617 Delta) from patients. HeLa-ACE2 cells infected at MOI 0.5–2 for 2 h, then medium replaced; harvested at 24 h post-infection. - Readouts: Western blot of cell lysates for SARS-CoV-2 nucleoprotein (NP) levels; actin controls. qRT-PCR for RNA quantification. Viral titers validated by cytopathic effect and Reed–Muench method. - Off-target and metagenomic checks: Host transcriptome sequencing quality (average Q30 >85%; read length >140 bp) and Kraken2 mapping to confirm minimal non-target coronavirus sequences. - Phylogenetics and coverage analysis: Local BLAST, MAFFT for MSA, MEGA X neighbor-joining phylogenies, and R (ggplot2, ggtree) to visualize mutation frequencies and crRNA coverage across SARS-CoV-2 lineages and other coronaviruses. - Top guides taken forward: crRNA-N18 and crRNA-3615 (N), and crRNA-1496, crRNA-1515, crRNA-1617, crRNA-1881 (ORF1ab/NSP13/NSP14).

- In vitro degradation: crRNA-N18 and crRNA-3615 targeting N reduced synthetic N fragments by >99%; crRNA-1496, -1515, -1617, and -1881 targeting ORF1ab achieved >99% reduction of ORF1ab fragments (qRT-PCR). - Infected cell models: Cas13d with pooled crRNAs suppressed SARS-CoV-2 replication in HeLa-ACE2 cells at MOIs up to 2, as shown by reduced NP protein levels on western blots. - Variant coverage in cells: The Cas13d–crRNA system suppressed ancestral (614D), B.1, Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617) infections; inhibition of Beta appeared marginally more effective than others. - Bioinformatic coverage of global genomes (n=1,651,448 sequences): • crRNA-1496: 1,651,447/1,651,448 (99.9999%) targeted • crRNA-1515: 1,651,443/1,651,448 (99.9997%) targeted • crRNA-1617: 1,651,444/1,651,448 (99.9998%) targeted • crRNA-1881: 1,651,448/1,651,448 (100.0%) targeted • crRNA-3615: 1,651,444/1,651,448 (99.9998%) targeted • crRNA-N18: 1,651,448/1,651,448 (100.0%) targeted - Omicron analysis: Predicted to be targetable by all six crRNAs with 0–1 mismatches. For Omicron sequences analyzed, five guides (N18, 1496, 1515, 1617, 3615) showed 100% with 0 mismatches; for crRNA-1881, 98.48% had 1 mismatch and 1.52% had 0 mismatches. - Specificity: Transcriptome quality metrics and Kraken2 mapping indicated minimal off-target degradation of host RNAs and negligible non-target coronavirus sequences. - Breadth: The six crRNAs specifically target the SARS(-like) coronavirus family; Middle East respiratory syndrome coronavirus (MERS-CoV) was not covered by these guides.

This study addresses whether CRISPR-Cas13d can be rapidly programmed to degrade authentic SARS-CoV-2 RNAs across major VOCs. By selecting conserved targets (NSP13, NSP14, N) and validating in both in vitro assays and infected human cells, the Cas13d–crRNA system achieved robust knockdown (>99% in vitro; marked NP reduction in cells) and suppressed replication of multiple lineages, including Delta and Alpha/Beta, with bioinformatic support for Omicron coverage. The results support Cas13d as a mutation-flexible antiviral platform: crRNAs are easily redesignable to track viral evolution, and the small Cas13d size facilitates delivery (AAV/LNP). Compared with prior Cas13a/b studies, this work provides evidence using authentic SARS-CoV-2 infections and demonstrates broad variant coverage with a minimal guide set. The near-complete in silico coverage of >1.65 million genomes suggests potential for pan-SARS-CoV-2 applications and a foundation for rapid response to future VOCs.

A reprogrammed CRISPR-Cas13d platform targeting conserved SARS-CoV-2 RNAs (NSP13, NSP14, N) effectively degrades viral RNA and inhibits replication of ancestral and VOC strains in human cells. Six optimized crRNAs provided near-universal genomic coverage, including predicted compatibility with Omicron, while maintaining specificity. Given Cas13d’s compact size and ease of guide redesign, this approach represents a rapid, adaptable antiviral strategy against current and emergent RNA virus threats. Future work should evaluate in vivo efficacy and delivery, expand guide sets for broader coronavirus coverage, and assess clinical feasibility.

- Experiments were conducted in cell culture (HeLa-ACE2) with authentic virus; no in vivo animal or clinical data were presented. - Efficacy was primarily measured by NP protein reduction and qRT-PCR; comprehensive viral titration across conditions was limited. - Bioinformatic coverage indicates predicted targeting of Omicron; direct experimental validation against Omicron in infected cells was not reported in detail. - The six-guide panel did not cover MERS-CoV; broader pan-coronavirus coverage would require additional guides. - Off-target assessments relied on sequencing quality metrics and metagenomic checks; deeper transcriptome-wide off-target cleavage analyses could strengthen specificity claims.