GRNA library targeting SARS-CoV-2 virus positive strand and negative strand subgenomes

Abstract

The invention belongs to the technical field of gene engineering, and relates to a gRNA library of a targeted SARS-CoV-2 virus subgenome. The library is obtained through multi-step rational design screening, is composed of 65 gRNAs, can specifically recognize and target positive chain and negative chain subgenome RNAs of viruses ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N and ORF10, and has a sequence as shown in SEQ ID NO: 1 to 65. The gRNA can form a CRISPR-Cas13b (clustered regularly interspaced short palindromic repeats-associated protein 13b) system with a Cas13b protein, and is used for non-therapeutically inhibiting the replication of the SARS-CoV-2 virus.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a gRNA library based on the CRISPR / Cas13b system, which systematically targets the positive and negative subgenomes of the SARS-CoV-2 virus. Background Technology

[0002] COVID-19, caused by SARS-CoV-2, poses a serious threat to public health. This virus is a single-stranded positive-sense RNA virus with a genome approximately 29.9 kb in length. In addition to the ORF1a / 1b region encoding the replicase, it contains open reading frames encoding various structural proteins (S, M, E, N) and accessory proteins (ORF3a, ORF6, ORF7a, etc.).

[0003] Studies on viral infection mechanisms have shown that the positive-sense genomic RNA of SARS-CoV-2 can serve as a template in the host cell to synthesize a complete complementary negative-sense RNA as a replication intermediate. At the same time, through a discontinuous transcription mechanism, a series of negative-sense subgenomic RNAs (-sgRNAs) of varying lengths are synthesized as templates, which are then transcribed to generate various positive-sense subgenomic mRNAs (+sgRNAs) with highly conserved 5' and 3' ends. These +sgRNAs are eventually translated into various viral structural proteins and accessory proteins.

[0004] The CRISPR-Cas system, particularly the RNA-targeting CRISPR-Cas13b system, can achieve antiviral effects by designing specific guide RNAs (gRNAs) to guide Cas13 effector proteins to specifically recognize and cleave viral RNA. Accordingly, a highly efficient antiviral strategy involves: targeting the +sgRNA used for viral protein translation to directly eliminate existing translation templates and inhibit viral protein synthesis; and targeting the -sgRNA, which serves as its transcription template, to intervene at the source of replication and transcription, inhibiting the generation of new viral mRNA.

[0005] Therefore, developing a gRNA library that can fully cover both the positive and negative strands of the viral genome is extremely important for the CRISPR / Cas13 system to function effectively and has significant application value. Summary of the Invention

[0006] The purpose of this invention is to provide a set of gRNA sequences targeting the positive and negative subgenomes of SARS-CoV2 virus, compositions containing such sequences, and their applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a gRNA library targeting SARSCoV2, which is constructed using the following method: Target selection: Using the SARS-CoV-2 reference genome (NC_045512.2) as a template, candidate gRNA sequences were designed for the positive and negative strand coding regions of the nine subgenomic units ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N and ORF10, respectively, to ensure that the library can fully cover the viral transcripts and their synthesis templates.

[0008] Multi-step screening: The above candidate sequences were subjected to the following steps in sequence: 1. Filtering sequences containing more than 4 consecutive T or C sequences to ensure transcription efficiency; 2. Ensuring target accessibility through secondary structure prediction; 3. Performing rigorous comparison with the human transcriptome to reduce off-target risk.

[0009] Library composition: After the above process, a core library consisting of 65 gRNAs is finally obtained, of which 32 target positive-sense subgenomic RNAs and 33 target negative-sense subgenomic RNAs.

[0010] In one embodiment, the gRNA and Cas13b protein together form a CRISPR system; cell experiments using this system have shown that it can reduce the expression level of virus-specific genes in SARS-CoV-2 replicons, thereby confirming the targeting activity of the gRNA of the present invention.

[0011] The beneficial effects of this invention include: Comprehensive targeting strategy: The gRNA library established in this invention can cover both the positive and negative strands targeting the SARS-CoV-2 subgenome.

[0012] Rigorous screening criteria: Multi-step screening ensures the stability, accessibility, and low off-target risk of gRNA. Attached Figure Description

[0013] Figure 1 This is a map showing the target distribution of the gRNA library on the positive and negative strand subgenomes of SARS-CoV-2.

[0014] Figure 2 RT-qPCR results for gRNA targeting SARS-CoV-2 ORF10. Detailed Implementation

[0015] Example 1: Screening and synthesis of gRNA libraries: Using the SARSCoV2 reference genome (NC_045512.2) as a template, candidate gRNA sequences covering the full length of each gene were designed for the positive and negative strands of nine subgenomic units: ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, and ORF10, and an initial sequence library was constructed.

[0016] The initial sequence library was subjected to three rounds of screening: Sequence-based screening: Filter all gRNA sequences containing >4 consecutive Ts or >4 consecutive Cs to ensure transcription efficiency and stability.

[0017] Secondary structure screening: The minimum free energy of the spacer region of each gRNA was calculated using the RNAfold online tool, and the sequence with the minimum free energy closest to 0 kcal / mol was selected to ensure that it is in an open conformation and can easily bind to the target RNA.

[0018] Off-target risk assessment: Candidate gRNAs are rigorously sequence-aligned with the human transcriptome to exclude sequences with significant homology to human genes, in order to minimize potential off-target risks.

[0019] When finally selecting the gRNA, the principle of uniformly covering each target gene region was followed to ensure the comprehensiveness of the library.

[0020] This process yielded a core library consisting of 65 highly specific gRNAs, the sequence information of which is shown in Table 1. A schematic diagram of the target site distribution on the viral genome is shown below. Figure 1 Of these, 32 targeted positive-sense subgenomic RNAs and 33 targeted negative-sense subgenomic RNAs. Thus, a comprehensive and rationally designed antiviral CRISPR gRNA library was successfully constructed.

[0021] Table 1 gRNA Sequence Name Sequence (5'→ 3') (+) ORF3a gRNA1 TGCAGTAGCGCGAACAAAATCTGAAGGAGTAGCATCC (+) ORF3a gRNA2 ACAGCAAGTTGCAAACAAAGTGAACACCCTTGGAGAGTG (+) ORF3a gRNA3 AGCAAAGAAAATAGTTGGCATCATAAAGTAATGGGTT (+) ORF3a gRNA4 AAGGTAACATGTTCAACACCAGTGTCTGTACTCAATTGA (+) ORF3a gRNA5 GCTAGTAGTCGTCGTCGGTTCATCATAAATTGGTTCCATT (+) E gRNA1 TATTAACTATTAACGTACCTGTCTCTTCCGAAACGAATGA (+) E gRNA2 GTTTTACAAGACTCACGTTAACAATATTGCAGCAGTACGC (+) E gRNA3 TTAGACCAGAAGATCAGGAACTCTAGAAGAATTCAGA (+) M gRNA1 AAAATTAACTTAATTATATACAAAAACCTATTCCTGTTGG (+) M gRNA2 CGTACGCGCAAACAGTCTGAAAGAAGCAATGAAGTAGCT (+) M gRNA3 CGAAGCTCCCAATTTGTAATAAGAAAGCGTTCGTGATGT (+) M gRNA4 TGTCACTTACTGTACAAGCAAAGCAATATTGTCACTGC (+) ORF6 gRNA1 AATATCTCTGCTATAGTAACCTGAAAGTCAACGAGATGAA (+) ORF6 gRNA2 TTTAATTATGAGGTTTATGATGTAATCAAGATTCCAAATG (+) ORF6 gRNA3 CCATTGGTTGCTCTTCATCTAATTGAGAATATTTATTCT (+) ORF7a gRNA1 ACAAGTAGCGAGTGTTATCAGTGCCAAGAAAAGAATAA (+) ORF7a gRNA2 GCAAGTCAGTGCAAATTTGTTATCAGCTAGAGGATGAAAT (+) ORF7a gRNA3 GTTTAGGTGAAACTGATCTGGCACGTAACTGATAGACGT (+) ORF7a gRNA4 ATTGCCGCAACAATAAGAAAAATTGGAGAGTAAAGTTCTT (+)ORF7b gRNA1 GCACAAATAGAAGTCAATTAATGAAAGTTCAATCATTCT (+)ORF7b gRNA2 TTCAAGTGAGAACCACAAGATAATAAGCATAATTAAAAC (+)ORF8 gRNA1 TGTAAACTACATTCTTGGTGAAATGCAGCTACAGTTG (+)ORF8 gRNA2 ATTTAGAACCAGCCTCATCCACGCACAATTCAATTAAAGG (+)ORF8 gRNA3 AGGTTCCTGGCAATTAATTGTAAAAGGTAAACAGGAAACT (+)ORF8 gRNA4 CTAAACAACACGAACGTCATGATACTCTAAAAGTCTTC (+)N gRNA1 AACGCCTTGTCCTCGAGGGAAT (+)N gRNA2 AGGCTCCCTCAGTTGCAACCCA (+)N gRNA3 GCCTCAGCAGCAGATTTCTTAG (+)N gRNA4 CTTGGGTTTGTTCTGGACCACG (+)N gRNA5 GAATGTTTTTGTATGCGTCAATA (+)ORF10 gRNA1 CATTCTGCACAAGAGTAGACTATATATCGTAAACGGAAAAGCG (+)ORF10 gRNA2 GATTGCTATGTGAGATTAAAGTTAACTACATCTACTTG (-)ORF3a gRNA1 ATGGAACCAATTTATGATGAACCGACGACGACTACTAGCG (-)ORF3a gRNA2 GTTGTATTACACAGTTACTTCACTTCAGACTATTACCAGC (-)ORF3a gRNA3 GAAATGCCGTTCCAAACCCATTACTTTATGATGCCAAC (-)ORF3a gRNA4 TCAGAGCGCTTCCAAAATCATAACCCTCAAAAGAGATGG (-)ORF3a gRNA5 TGTTTATGAGAATCTTCACAATTGGAACTGTAACTTTGAA (-)E gRNA1 CGTTTACTCTCGTGTTAAAAATCTGAATTCTTCTAGAGTT (-)E gRNA2 CGTACTGCTGCAATATTGTTAACGTGAGTCTTGTAAAACC (-)E gRNA3 ATTCGTTTCGGAAGAGACAGGTACGTTAATAGTTAATAGC (-)M gRNA1 ATTGGCAACTATAAATTAAACACAGACCATTCCAGTAGCA (-)M gRNA2 CTGTTGCTACATCACGAACGCTTTCTTATTACAAATTGGG (-)M gRNA3 TTCAATCCAGAAACTAACATTCTTCTCAACGTGCCACTCC (-)M gRNA4 TTGTGCTTGCTGCTGTTTACAGAATAAATTGGATCACCGG (-)M gRNA5 TGGATTTGTCTTCTACAATTTGCCTATGCCAAACAGGAATA (-)ORF6 gRNA1 GAATAAATATTCTCAATAGATGAAGAGCAACCAATGGAG (-)ORF6 gRNA2 TGATTACATCATAAACCTCATAATTAAAAATTTATCTAAG (-)ORF6 gRNA3 TCAGGTTACTATAGCAGAGATATTACTAATTATTATGAGG (-)ORF7a gRNA1 TGTTTATAACACTTTGCTTCACACTCAAAGAAAGACAGA (-)ORF7a gRNA2 ACGTCTATCAGTTACGTGCCAGATCAGTTTCACCTAAACT (-)ORF7a gRNA3 GGCAATTCACCATTTCATCCTCTAGCTGATAACAAATTTG (-)ORF7a gRNA4 GCTACTTGTGAGCTTTATCACTACCAAGAGTGTGTTAGAG (-)ORF7b gRNA1 AAGATCATAATGAAACTTGTCACGCCTAAACGAACATGAA (-)ORF7b gRNA2 TGATTGAACTTTCATTAATTGACTTCTATTTGTGCTTT (-)ORF8 gRNA1 GGAACCTAAATTGGGTAGTCTTGTAGTGCGTTGTTCGTTC (-)ORF8 gRNA2 CCCATTCAGTACATCGATATCGGTAATTATACAGTTTCCT (-)ORF8 gRNA3 ACCCGTGTCCTATTCACTTCTATTCTAAATGGTATATTAG (-)ORF8 gRNA4 TAGGAATCATCACAACTGTAGCTGCATTTCACCAAGAATG (-)N gRNA1 AACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAA (-)N gRNA2 ACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGA (-)N gRNA3 AAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAA (-)N gRNA4 GCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAA (-)N gRNA5 TCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCG (-)ORF10 gRNA1 ACATAGCACAAGTAGATGTAGTTAACTTTAATCTCACATA (-)ORF10 gRNA2 TTTACGATATATAGTCTACTCTTGTGCAGAATGAATTCTC Example 2: gRNA targeting validation: Based on the CRISPR-Cas13b system principle, this embodiment selected gRNA-2, which targets SARS-CoV-2 (+)ORF10sgRNA, and gRNA-1, which targets (-)ORF10sgRNA, respectively, with non-targeting gRNA (NT-gRNA) as a control, and verified the gRNA targeting by RT-qPCR.

[0022] 1. Experimental Materials and Methods: HEK293T cell lines were either preserved in our laboratory or sourced from other routine laboratories and cell banks. Fetal bovine serum was purchased from Gbico. Cell transfection reagents were purchased from Lipo8000™ at Beyotime Biotechnology Co., Ltd. Evo M-MLV reverse transcriptase and 2×SYBR Green Pro Tap HS kits were purchased from Hunan Aikerui Biotechnology Co., Ltd. The real-time PCR instrument (CFX96) was a product of Bio-Rad Laboratories, USA.

[0023] 2. Experimental Methods: 2.1 Cell transfection: HEK293T cells were seeded into 6-well plates at a predetermined amount and cultured overnight until the cells adhered and reached 60%-70% confluence. Transfection was then performed using Lipo8000™ transfection reagent. SARS-CoV-2 replicons, plasmids expressing Cas13b, and plasmids expressing gRNA were co-transfected into the cells at a mass ratio of 2:1.5:1 (total plasmid amount 4.5 μg per well). Cells were incubated at 37°C in a CO2 incubator for 4–6 hours. The medium containing the DNA-Lipo8000 mixture was then removed, and the medium was replaced with fresh cell culture medium containing 10% FBS for another 48 hours.

[0024] 2.2 Total RNA extraction from cells and RT-qPCR detection of target mRNA: Total RNA was extracted from cells using Trizol reagent, following the reagent's standard instructions. The extracted RNA was reverse transcribed using Evo M-MLV reverse transcriptase at 37℃ for 15 min to obtain cDNA. RT-qPCR analysis was then performed according to the instructions for the 2×SYBR Green Pro Tap HS kit. Primers used are shown in Table 2. The initial temperature was set at 65℃; temperature variations were set at 0.5℃; and the final temperature was set at 95℃. After the RT-qPCR reaction, amplification and melting curves were confirmed, and the results were statistically analyzed using the ΔΔCt method. Where ΔCt = target gene Ct value - internal reference gene Ct value, ΔΔCt = experimental group ΔCt - control group ΔCt, and relative gene expression level = 2. -ΔΔCt .

[0025] Table 2 RT-qPCR primer sequences Name Sequence (5’→3’) q-GAPDH-F AGCCACATCGCTCAGACAC q-GAPDH-R GCCCAATACGACCAAATCC q-CmR-F ACCGTAACACGCCACATCTT q-CmR-R TTCTTGCCCGCCTGATGAAT q-N-F ACATTCCCACCAACAGAGCC q-N-R CAGCACTGCTCATGGATTG 3. Experimental Results: The results are as follows Figure 2As shown, compared with the control group transfected with NT-gRNA, the experimental groups transfected with gRNA-1 and gRNA-2 showed reduced mRNA expression levels of SARS-CoV-2 ORF10. This result indicates that both gRNAs, targeting the positive and negative strands of the virus respectively, can effectively guide the Cas13b system to cleave the target sequence, validating their targeting activity.

Claims

1. A gRNA combination targeting a SARS CoV2 viral subgenomic, characterized in that, The combination comprises a plurality of gRNAs capable of specifically targeting the positive and negative subgenomic RNAs of SARS-CoV2 virus corresponding to the ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N and ORF10 genes.

2. The gRNA combination of claim 1, wherein, The gRNA combination consists of 32 gRNAs targeting the positive subgenomic RNA and 33 gRNAs targeting the negative subgenomic RNA.

3. The gRNA combination of claim 2, wherein, The nucleotide sequence of the gRNA is shown in SEQ ID NO: 1 to SEQ ID NO:

65.

4. A CRISPR Casl3b system, characterized in that A CRISPR Cas13b system comprising the gRNA combination of any one of claims 1 to 3 and a Cas13b protein.

5. Use of the gRNA combination of any one of claims 1 to 3 or the CRISPR Cas13b system of claim 4 in the manufacture of a kit for detecting SARS-CoV2 viral RNA.

6. A method of non-therapeutically inhibiting replication of SARS CoV2 virus, characterized in that, The method comprises introducing the CRISPR Cas13b system of claim 4 into a cell and contacting the system with the viral RNA.

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