Method for screening sgRNA high-efficiency action target based on CRISPR-Cas13d system and application
阅读说明：本技术 基于CRISPR-Cas13d系统的sgRNA高效作用靶点的筛选方法及应用 (Method for screening sgRNA high-efficiency action target based on CRISPR-Cas13d system and application ) 是由 胡晓湘 李果 王鑫杰 李宁 于 2019-10-12 设计创作，主要内容包括：本发明提供一种基于CRISPR-Cas13d系统的sgRNA高效作用靶点的筛选方法及应用,特别是在RNA病毒敲降方面的应用。本发明利用mCherry荧光报告基因,将PRRSV病毒ORF4和ORF5两个表达阅读框序列分别融合到mCherry基因的碳端构建筛选CRISPR-Cas13d系统的sgRNA高效作用靶点的荧光报告系统,利用CRISPR-Cas13d高效切割mRNA的特性,进行高效率sgRNAs的快速筛选。然后利用筛选出的高效靶向结合的sgRNAs进行CRISPR-Cas13d系统高效降解PRRSV-GFP重组病毒。本发明提供的RNA病毒敲降方法具有高效率,高精准率及低脱靶率的优势。(The invention provides a screening method and application of a sgRNA high-efficiency acting target based on a CRISPR-Cas13d system, in particular to application in RNA virus knockdown. The invention utilizes mCherry fluorescent reporter gene to respectively fuse two expression reading frame sequences of PRRSV ORF4 and ORF5 to the carbon end of the mChery gene to construct a fluorescent reporter system for screening the sgRNA high-efficiency action target of a CRISPR-Cas13d system, and utilizes the characteristic of CRISPR-Cas13d high-efficiency cutting of mRNA to rapidly screen high-efficiency sgRNAs. And then carrying out CRISPR-Cas13d system high-efficiency degradation on the PRRSV-GFP recombinant virus by using the screened high-efficiency targeting combined sgRNAs. The RNA virus knockdown method provided by the invention has the advantages of high efficiency, high precision and low off-target rate.)
1. The method for screening the sgRNA high-efficiency acting target based on the CRISPR-Cas13d system is characterized by comprising the following steps of:
1) synthesizing a target nucleic acid sequence, constructing the target nucleic acid sequence into a vector containing a report group, and positioning the target nucleic acid sequence and the report group in the same expression cassette to obtain a report vector;
2) designing and synthesizing a series of sgRNA sequences based on a CRISPR-Cas13d system according to a target nucleic acid sequence, and respectively constructing the sgRNA sequences into sgRNA expression vectors based on the CRISPR-Cas13d system;
3) introducing the report vector of the step 1), an expression vector containing Cas13d protein and the recombinant vector obtained in the step 2) into eukaryotic cells together to obtain a series of transformed cells; after the transformed cells are cultured for a period of time, respectively identifying the cutting efficiency of different sgRNAs on a target nucleic acid sequence, and screening out high-efficiency sgRNAs according to the cutting efficiency;
wherein, the target nucleic acid sequence in the step 1) contains or encodes a nucleotide sequence with a cap structure at the 5 'end and a poly-adenine nucleotide tail structure at the 3' end.
2. The method of claim 1, wherein the nucleic acid sequence of interest is an mRNA or RNA viral nucleic acid sequence;
preferably, the RNA virus is PRRSV.
3. The method of claim 1, wherein the reporter group-containing vector is a eukaryotic expression vector carrying mCherry fluorescent protein;
preferably, the target nucleic acid sequence is located at the C-terminus of the mCherry fluorescent protein.
4. The method according to claim 1, characterized in that the sgRNA expression vector of step 2) based on the CRISPR-Cas13d system is pC0043-PspCas13b crRNA backbone plasmid.
5. The method of claim 1, wherein the expression vector containing Cas13d protein in step 3) is a vector with NES or NLS sequence linked at two ends of Cas13d protein;
preferably, the expression vector containing the Cas13d protein has a sequence as shown in SEQ ID NO 3 or 4.
6. The method of any one of claims 1 to 5, wherein the target nucleic acid sequence is the ORF4 and/or ORF5 gene of PRRSV; the nucleic acid sequences of the action sites of the high-efficiency sgRNAs screened aiming at the ORF4 and ORF5 genes are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6; and/or
The eukaryotic cell in the step 3) is HEK 293T.
7. Use of a high-efficiency sgRNA obtained by the method according to any one of claims 1 to 6 for knockdown of a non-therapeutic RNA virus of interest based on the CRISPR-Cas13d system.
8. The application according to claim 7, wherein the application comprises: (1) selecting a target nucleic acid sequence according to an RNA virus to be knocked down, and screening out the high-efficiency sgRNA by using the method of any one of claims 1 to 6; (2) constructing a high-efficiency sgRNA sequence into a sgRNA expression vector based on a CRISPR-Cas13d system, and introducing the obtained recombinant vector and an expression vector containing Cas13d protein into a eukaryotic host cell infected with the RNA virus to be knocked down.
9. The use of claim 8, wherein the RNA virus is PRRSV and the target nucleic acid sequence is ORF4 and/or ORF5 gene of PRRSV; the nucleic acid sequences of the action sites of the high-efficiency sgRNAs screened aiming at the ORF4 and ORF5 genes are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6; and/or
The expression vector containing the Cas13d protein is a vector with NES sequences connected at two ends of the Cas13d protein, and the sequences are shown as SEQ ID NO. 3.
10. A kit for knock-down of an RNA virus, comprising: a high efficiency sgRNA obtained according to the method of any one of claims 1-6, and optionally including an expression vector comprising a Cas13d protein.
Technical Field
The invention relates to the technical field of gene editing, in particular to a method for screening sgRNA high-efficiency acting targets based on a CRISPR-Cas13d system and application thereof.
Background
RNA virus is a virus which seriously harms human health and is produced safely in agriculture. Because of its characteristics of high degree of variation, rapid replication, complex life activities, etc., prevention and control of RNA viruses and treatment of related diseases become particularly difficult. The main prevention and treatment methods for common RNA viruses and related diseases are the research and development of vaccines and antibodies and RNA interference technology (RNAi), but the effects are not ideal, and the exploration and development of new methods are imminent.
In recent years, the programmable nuclease-mediated gene editing technology is rapidly developed, and the CRISPR (clustered regulated interleaved short palindromic repeats) system gene editing technology is widely applied to the research fields of biology, basic medicine and the like, so that the technology is fully proved to have huge development potential. The CRISPR system is part of the immune defense mechanism of bacteria and archaea, and after foreign plasmids or viruses invade a host, the CRISPR recognizes these foreign DNA through internal spacer sequences and forms memory. When the phage invades again, a sequence in the CRISPR region is transcribed into RNA (pre-crRNA) of a precursor, the pre-crRNAs are transcriptionally activated by transcription activating RNA (tracrrna) to become small mature crRNA, and bind to the associated Cas protein to form a crRNA-Cas protein complex, and then precisely bind to the target DNA through base complementary pairing, so that the Cas protein is guided to cut the target DNA, and target DNA Double Strand Breaks (DSBs) are caused by the endonucleases. Repair via the non-homologous end joining (NHEJ) pathway can result in non-specific base deletions, insertions, or other forms of mutation; DSBs can also use DNA repair templates, such as single-stranded oligonucleotides (ssODN) that correct mutations or insert new gene sequences at the cleavage site via homologous recombination repair (HDR) [ Hsu et al, 2014; kim and Kim, 2014; komor et al, 2017 ]. The type II CRISPR system acting with Cas9 protein is widely used in gene editing in mammals due to no need of complex protein complex and simple system composition [ Barrangou R & Doudna JA, 2016; komor et al, 2017 ].
Recent studies have shown that class ii type VI CRISPR effector protein Cas13d proteins cleave ssRNA with high efficiency (silverana Konermann et al, 2018). Rfx-Cas13d belongs to the CRISPR-Cas
Disclosure of Invention
The invention aims to provide a screening method and application of a sgRNA high-efficiency action target based on a CRISPR-Cas13d system.
The inventors provide a knock-down strategy for RNA viruses based on the high efficiency, precision and specificity of CRISPR-Cas13d system-mediated ssRNA cleavage, in particular Rfx-Cas13d (CasRx) -mediated ssRNA cleavage: efficient sgRNAs are screened by cutting mCherry-ORF4/ORF5 report plasmid mRNA through a CRISPR-Cas13d system, the screened efficient sgRNAs are used for efficient knockdown of PRRSV-GFP recombinant RNA viruses, and replication of the RNA viruses is inhibited, so that prevention and treatment of the RNA viruses are realized.
In a first aspect, the invention provides a method for screening a sgRNA high-efficiency acting target based on a CRISPR-Cas13d system, which comprises the following steps:
1) synthesizing a target nucleic acid sequence, constructing the target nucleic acid sequence into a vector containing a report group, and positioning the target nucleic acid sequence and the report group in the same expression cassette to obtain a report vector;
2) designing and synthesizing a series of sgRNA sequences based on a CRISPR-Cas13d system according to a target nucleic acid sequence, and respectively constructing the sgRNA sequences into sgRNA expression vectors based on the CRISPR-Cas13d system;
3) introducing the report vector of the step 1), an expression vector containing Cas13d protein and the recombinant vector obtained in the step 2) into eukaryotic cells together to obtain a series of transformed cells; after the transformed cells are cultured for a period of time, respectively identifying the cutting efficiency of different sgRNAs on a target nucleic acid sequence, and screening out high-efficiency sgRNAs according to the cutting efficiency;
wherein, the target nucleic acid sequence in the step 1) contains or encodes a nucleotide sequence with a cap structure at the 5 'end and a poly-adenine nucleotide tail structure at the 3' end.
The target nucleic acid sequence includes, but is not limited to, an mRNA or RNA viral nucleic acid sequence.
Preferably, the RNA virus is PRRSV (porcine reproductive and respiratory syndrome virus).
The vector containing the reporter group is a eukaryotic expression vector carrying mCherry fluorescent protein, such as pcDNA3.1-mCherry.
In one embodiment of the present invention, the reporter vector is constructed as follows: ORF4/ORF5 sequences obtained by amplification of PRRSV virus cDNA are connected to the downstream of mCherry gene in commonly used commercial vector pcDNA3.1-mCherry by utilizing a vector homologous recombination method, and mCherry-ORF4 and mChery-ORF 5 fluorescent protein reporter vectors are respectively constructed.
Preferably, the target nucleic acid sequence is located at the C-terminus of the mCherry fluorescent protein.
The sgRNA expression vector based on the CRISPR-Cas13D system described in the preceding method, step 2) was the pC0043-PspCas13b crRNA backbone mammalian gene editing plasmid (see silverana Konermann et. transcriptome Engineering with RNA-Targeting Type VI-D CRISPR effects. (2018). Cell).
The method comprises the step 3) that the expression vector containing the Cas13d protein is a vector with NES or NLS sequences connected to both ends of the Cas13d protein.
Preferably, the expression vector containing the Cas13d protein has a sequence as shown in SEQ ID NO 3 or 4.
More preferably, the target nucleic acid sequence is the ORF4 and/or ORF5 gene of PRRSV; the nucleic acid sequences of the action sites of the high-efficiency sgRNAs screened by aiming at the ORF4 and ORF5 genes are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6.
Optionally, the eukaryotic cell of step 3) is HEK 293T.
In a second aspect, the invention provides the use of the high efficiency sgRNA obtained according to the above method for (non-therapeutic-purpose) RNA virus knockdown based on the CRISPR-Cas13d system.
The application comprises the following steps: (1) selecting a target nucleic acid sequence according to an RNA virus to be knocked down, and screening out the efficient sgRNA by using the method; (2) constructing a high-efficiency sgRNA sequence into a sgRNA expression vector based on a CRISPR-Cas13d system, and introducing the obtained recombinant vector and an expression vector containing Cas13d protein into a eukaryotic host cell infected with the RNA virus to be knocked down.
In one embodiment of the invention, the RNA virus is PRRSV and the target nucleic acid sequence is the ORF4 and/or ORF5 gene of PRRSV; the nucleic acid sequences of the action sites of the high-efficiency sgRNAs screened by aiming at the ORF4 and ORF5 genes are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6.
Preferably, the expression vector containing the Cas13d protein is a vector with NES sequences connected to both ends of the Cas13d protein, and the sequences are shown as SEQ ID NO. 3.
In a third aspect, the present invention provides a kit for knock-down of an RNA virus, the kit comprising: a high efficiency sgRNA obtained according to the above method, and optionally an expression vector comprising a Cas13d protein.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention provides a screening tool for efficiently targeting sgRNA by a CRISPR-Cas13d system, and has universality. The invention fully utilizes the target sequence connected to the downstream of the mChery fluorescent gene to construct a fusion protein report system, shows the efficiency of targeting cutting mRNA by Cas13d through the change of the fluorescent value, screens the sgRNA in high-efficiency targeting combination, fuses two expression reading frame sequences of PRRSV ORF4 and ORF5 to the carbon end structure of the mChery gene respectively, selects a relatively conservative region to design the sgRNA through the comparison of different PRRSV variant sequences, and successfully screens two high-efficiency sgRNAs aiming at PRRSV ORs 4 and ORF5 by utilizing the mChery-ORF 4/ORF5 report system, and the screening method is suitable for the gRNA screening of all target mRNA degradation corresponding genes.
(II) high efficiency of degrading RNA virus. Efficient sgRNA obtained by screening through a fluorescence report system is combined with a CRISPR-Cas13d system, efficient degradation of PPRSV recombinant virus is successfully carried out on Marc145 cells, the titer of the cut virus is reduced, the expression quantity is reduced, the efficient inhibition of the method on RNA virus is fully displayed, and the method is suitable for inhibiting other RNA viruses.
And thirdly, the invention has the advantages of high accuracy and low miss ratio. The cell activity and the potential off-target site of the cell transfected by the CRISPR-Cas13d system are detected, and the experimental result shows that the cell activity state is not influenced and no off-target phenomenon exists, so that the superiority of the invention is further proved. The RNA virus knockdown method provided by the invention has the advantages of high efficiency, high precision and low off-target rate, and can be applied to the fields of agricultural animal disease resistance breeding, research on human RNA viral diseases, research and development of novel medicines and the like.
Drawings
Fig. 1 is a schematic diagram of efficient sgRNA screening achieved by cutting mCherry-ORF4(a) and mCherry-ORF5(B) with a CRISPR-Cas13d system in embodiment 1 of the present invention.
FIG. 2 is a schematic diagram of the method for cutting PRRSV-GFP recombinant RNA virus by using engineered NES-Cas13d (A) and NLS-Cas13d (B) in example 1 of the present invention to achieve high efficiency knockdown of RNA virus.
FIG. 3 is a graph showing the effect of mCherry-ORF4(A) and mCherry-ORF5(B) reporter systems on the knockdown of mRNA in HEK293T in example 1 of the present invention.
FIG. 4 is a graph showing the knockdown effect of PRRSV-GFP recombinant virus on Marc145 cells in example 2 of the present invention.
FIG. 5 is a diagram showing potential off-target site gene expression amount detection of CRISPR-Cas13d system knockdown PRRSV-GFP virus collecting cells in example 2 of the invention; a, B is respectively used for detecting the relative expression quantity OF mRNA OF an OF4-sgRNA1 targeted genome potential off-target gene NUP210 and MAGI1, compared with a control group, the expression quantity is not obviously changed, and no off-target condition occurs in an ORF4-sgRNA1 targeted group; C. d is the detection OF the mRNA relative expression quantity OF OF5-sgRNA1 targeted genome potential off-target genes F11-AS1 and CPEB2-AS1 respectively, compared with a control group, the expression quantity has no obvious change, and the off-target condition OF an ORF5-sgRNA1 targeted group appears.
Detailed Description
According to a first aspect of the present invention, there is provided a method for knock-down of an RNA virus, comprising:
the mCherry-ORF4/ORF5 fluorescent protein reporter vector was constructed and the 30bp template sequences (no PAM restriction) of the selected PRRSV virus relatively conserved coding region (CDS region) ORF4/ORF5 were aligned.
The sgRNA sequence was used to localize Cas13d to the target sequence so that the viral RNA was cleaved, resulting in knock-down of the viral RNA.
The sgRNA sequence is a 30bp sequence which is consistent with the target sequence in the forward direction.
According to the invention, the Cas13d protein carrier may be selected from: RfxCas13d-NLS-HA (CasRx). See Silvera Konermann et al Transcriptome Engineering with RNA-Targeting Type VI-D CRISPERFFEREctors (2018).
According to the method of the present invention, mRNA that can be used for knock-down is derived from the following three target genes: mCherry-ORF4, mCherry-ORF5 and PRRSV-GFP recombinant virus ORF4 and ORF5, and their corresponding sgRNA sequences are identical to 30bp of the selected target gene sequence.
According to a second aspect of the invention, the above method is provided for screening sgRNAs for CRISPR-Cas13d high efficiency cleavage of mRNA in cell line HEK293T in mCherry-ORF4/ORF5 reporter system.
According to the third aspect of the invention, the application of the method in the CRISPR-Cas13d system-mediated efficient knockdown of the PRRSV-GFP recombinant RNA virus in the cell line Marc145 is provided.
According to a fourth aspect of the present invention there is provided an isolated Marc145 cell line or subculture thereof obtained according to the above application.
According to a fifth aspect of the present invention, there is provided a kit for RNA virus knockdown, comprising a selected high-efficiency sgRNA, an engineered Cas13d vector, and amplification reagents.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions. Example 1 screening of sgRNA high-efficiency target for PRRSV based on CRISPR-Cas13d system
Firstly, constructing mCherry-ORF4/ORF5 fluorescence report system
As shown in FIG. 1, ORF4/ORF5 sequences were amplified and fused to the carbon end (C-end) of the mCherry gene to form the following two fluorescent reporter systems:
(1) mCherry-ORF4, FIG. 1(A), SEQ ID NO: 1;
(2) mCherry-ORF5, FIG. 1(B), SEQ ID NO: 2;
the construction method of the report vector comprises the following steps: ORF4/ORF5 sequences obtained by amplification of PRRSV virus cDNA are connected to the downstream of mCherry gene in commonly used commercial vector pcDNA3.1-mCherry by utilizing a vector homologous recombination method, and mCherry-ORF4 and mChery-ORF 5 fluorescent protein reporter vectors are respectively constructed.
Simultaneously, a nuclear signal NES and nuclear signal NLS mediated Cas13d system is constructed, as shown in FIG. 2, synthesized NES and NLS sequences are respectively inserted into the head and tail ends of Cas13d protein, and the following two engineered Cas13d expression systems are formed:
(1) NES-Cas13d-NES, FIG. 2(A), SEQ ID NO: 3;
(2) NLS-Cas13d-NLS, FIG. 2(B), SEQ ID NO: 4;
design of sgrnas was then performed. The Cas13d protease has no requirement for flanking sequences of interest and can target any RNA sequence. The sgRNA is selected and designed as follows:
through comparing different strain sequences of 10 PRRSV, selecting a relatively conservative region in ORF4 and ORF5 open reading frames to design sgRNAs, wherein the lengths of the sgRNAs are both 30bp, and respectively designing 3 pairs of sgRNAs for efficiency verification;
a 30bp sgRNA sequence was prepared in direct alignment with the target sequence.
The invention selects the following target sequences to design the corresponding sgrnas:
PRRSV-ORF4：
ORF4-sgRNA1:GATGTCCGAAAGACTCGAACTGAAACATGG
ORF4-sgRNA2:CGGCACTGAGAACTTTTGCGAATCGTCGGA
ORF4-sgRNA3:ATGTAGATAATTTTCATCTGTGACATTGGC
PRRSV-ORF5：
ORF5-sgRNA1:TGCTACTCAAGACATACCGCCCGTGATAAT
ORF5-sgRNA2:AGATGACAAAAGTCTCCACTGCCCAGTCAA
ORF5-sgRNA3:TGTGTCAAGGAAATGGCTGGTGGTGAGTGC
aiming at the selected target gene sequence, three ORF4 and three ORF5 construct corresponding sgRNA expression vectors, and different sgRNAs are respectively introduced into pC0043-PspCas13b crRNA backbone mammalian gene editing plasmids (sgRNA expression vectors).
Secondly, mGluR cutting of mGluerry-ORF 4/5 report plasmid mediated by CRISPR-Cas13d system is carried out on cell strain, and efficient targeted combination sgRNA screening is realized
mRNA knock-down (by electroporation or lipofection) of cell lines is performed as usual, taking lipofection as an example.
(1) Taking HEK293T cells as an example, the invention carries out the culture and transfection of eukaryotic cells: HEK293T cells were seeded in DMEM high-sugar medium (HyClone, SH30022.01B) supplemented with 10% FBS, containing penicillin (100U/ml) and streptomycin (100. mu.g/ml).
(2) The cells were divided into 24-well plates before transfection, and transfection was performed until the density reached 70% -80%.
(3) Transfection is exemplified by lipofection. According to LipofectamineTM2000 transformation Reagent (Invitrogen,11668-019) operation manual, taking mCherry-ORF4 reporter plasmid as an example, 50ng of mCherry-ORF4/mCherry-ORF5 plasmid, 300ng of NES-Cas13d-NES/NLS-Cas13d-NLS and 600ng of pC0043-PspCas13b crRNA backbone plasmid are mixed uniformly, and are transfected into each hole of cells, the solution is changed after 6-8 hours, and the cleavage efficiency is identified and detected after 48 hours.
(4) mRNA cleavage efficiency analysis
A. After transfecting the cells for 48h, washing the cells twice by using PBS, supplementing 500ul of a culture medium with 10% FBS concentration, performing cell fluorescence imaging photographing, and then digesting partial cells to perform C-FLOW detection on mCherry fluorescence efficiency;
B. collecting another part of cells, extracting total RNA by using a Trizol method, measuring the concentration of the extracted RNA, performing reverse transcription by using a reverse transcription reagent to obtain cDNA, and performing the reverse transcription by using a reverse transcription system (taking 1 mu g as an example):
mu.g mRNA, 4. mu.l 5 × HiScript II Select qRT SuperMix, 4. mu.l 4 × gDNA wiper Mix, RNase-free ddH2And O, the volume is filled to 20 mu l.
Reverse transcription program: 42 ℃ for 15 min; 20min at 50 ℃; 5s at 85 ℃; keeping at 4 ℃.
C. Real-time fluorescent quantitative PCR: Q-PCR detection primers are designed aiming at ORF4/ORF5, and Q-PCR is carried out by using a reverse transcription product (diluted by 10 times) to detect the relative expression quantity of mCherry-ORF4/ORF 5mRNA after 48h of transfection. The Q-PCR reaction system is as follows: mu.l cDNA, 5.
Q-PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 30s for 30 cycles; melting curve analysis was selected according to the instrument instructions: at 95 ℃ for 15s, at 60 ℃ for 15s, at 95 ℃ by△△Quantitative data were analyzed by CT method. The Q-PCR primers were as follows:
PRRSV-ORF4：
Forward:ATGGCTGCGTCCTTTCTTTTC
Reverse:GTCCTGGAGGACAACGAAGTC
PRRSV-ORF5：
Forward:ATGTTGGGGAAGTGCTTGACCGCG
Reverse:CGTTAAGTTATAAATCAACTG
GAPDH (internal reference gene):
Forward:AGAAGGCTGGGGCTCATTTG
Reverse:AGGGGCCATCCACAGTCTTC
the results show that mCherry-ORF4/5 reporter plasmid mRNA is knocked down with high efficiency up to 96%, wherein ORF4-sgRNA1(SEQ ID NO:5) and ORF5-sgRNA1(SEQ ID NO:6) have the best effect and the best biological repeatability, and the sgRNA is selected as the sgRNA knocked down by subsequent PRRSV-GFP recombinant viruses (FIG. 3, A and B). Example 2 knockdown of PRRSV-GFP recombinant viruses
An engineered NLS-/NES-Cas13d is designed to carry out the knockdown of PRRSV-GFP recombinant virus on Marc145 cells.
(1) Marc145 cells were seeded in DMEM high-sugar medium (HyClone, SH30022.01B) containing penicillin (100U/ml) and streptomycin (100. mu.g/ml) supplemented with 10% FBS.
(2) The cells were divided into 24-well plates before transfection, and transfection was performed until the density reached 70% -80%.
(3) Transfection is exemplified by lipofection. According to LipofectamineTM2000Transfection Reagent (Invitrogen, 11668-;
(4) RNA virus infection of cells: infection experiments with PRRSV-GFP recombinant RNA viruses were performed 12 hours after cell transfection (control titer MOI 0.01). Discarding cell culture solution, uniformly mixing virus and serum-free culture medium DMEM, adding the mixture into each well cell, culturing at 37 ℃ for 2h, supplementing 10% FBS complete culture medium, continuously culturing, collecting culture medium supernatant infected by the virus for 0h, 12h, 24h, 36h, 48h, 72h and other different time periods, and performing virus titer determination and virus mRNA expression level detection by using a real-time fluorescence quantitative PCR technology. Meanwhile, GFP fluorescence imaging, C-FLOW fluorescence rate detection and Q-PCR detection of the cells are carried out at 48h to detect the relative expression level of virus mRNA in the cells.
(5) mRNA cleavage efficiency analysis
A. After transfecting the cells for 48h, washing the cells twice by using PBS, supplementing 500ul of a culture medium with 10% FBS concentration, performing cell fluorescence imaging photographing, and then digesting partial cells to perform C-FLOW detection on mCherry fluorescence efficiency;
B. another part of the cells were extracted with total RNA by Trizol method, the concentration of the extracted RNA was measured, and reverse transcription was performed using a reverse transcription reagent to obtain cDNA, a reverse transcription system (1. mu.g as an example): mu.g mRNA, 4. mu.l 5 × HiScript IISelect qRT SuperMix, 4. mu.l 4 × gDNA wiper Mix, RNase-free ddH2And O, the volume is filled to 20 mu l.
Reverse transcription program: 42 ℃ for 15 min; 20min at 50 ℃; 5s at 85 ℃; keeping at 4 ℃.
C. Q-PCR was performed using the reverse transcription product (diluted 10-fold) to detect the relative expression of PRRSV-ORF4/ORF5mRNA 48h after transfection. The Q-PCR reaction system is as follows: mu.l cDNA, 5.
Q-PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 30s for 30 cycles; melting curve analysis was selected according to the instrument instructions: at 95 ℃ for 15s, at 60 ℃ for 15s, at 95 ℃ by△△Quantitative data were analyzed by CT method. The Q-PCR primers were as follows:
PRRSV-ORF4：
Forward:ATGGCTGCGTCCTTTCTTTTC
Reverse:GTCCTGGAGGACAACGAAGTC
PRRSV-ORF5：
Forward:ATGTTGGGGAAGTGCTTGACCGCG
Reverse:CGTTAAGTTATAAATCAACTG
GAPDH (internal reference gene):
Forward:AGAAGGCTGGGGCTCATTTG
Reverse:AGGGGCCATCCACAGTCTTC
(6) virus titer determination (TCID50 method)
And (3) within the time range of 0-72h when the transfected cells are infected with the PRRSV-GFP recombinant virus, respectively collecting cell culture solutions at 0h, 12h, 24h, 36h, 48h, 72h and other time points, and determining the virus titer by using a TCID50 method.
Determination of viral titer by TCID50 method:
A. cell preparation: after culturing Marc-145 cells for 24-48h, subculturing the Marc-145 cells into 96-well plates, and enabling the cells in each 96-well plate to be 2 x 10 by a cell counting method4100. mu.l per well, 5% CO at 37 ℃2The cells are grown in an incubator for 12-24 hours to 80-90% confluency before inoculation with virus.
B. Virus preparation: mu.l of each virus sample was added to 900. mu.l of DMEM maintenance medium containing 2% FBS and 1% PS, 100. mu.l of stock solution was diluted 10-fold, and 9-10 dilutions were set.
C. Cell inoculation: the cells were washed once with PBS, the supernatant was discarded, and 100. mu.l of virus solution was added in order of dilution from high to low, and 8 wells were inoculated for each gradient, taking care not to dry the cells. At least 5 control wells were also set without virus, and maintenance medium was added.
D. And (3) virus culture: is connected withPost-seeded Petri dishes at 37 ℃ with 5% CO2The cells were incubated in an incubator and observed daily for cytopathic loading and recorded.
E. And (3) calculating the titer: after 4-5 days of cell culture, the number of lesion wells per gradient was counted and the TCID50 value was calculated using the method of Reed-Muench.
(7) Q-PCR method for detecting relative expression quantity of RNA virus in cell culture solution
Total RNA extraction of cell culture solution is carried out at time points of 0h, 12h, 24h, 36h, 48h, 72h and the like by using a Trizol method, and cDNA inversion and Q-PCR are carried out according to the steps to quantitatively detect the relative expression quantity of PRRSV-ORF4/ORF 5.
(8) Activity assay of transfected cells
After the cells were transfected for 48h, the cell activity was detected using the MTT assay kit.
The knockdown effect of the PRRSV-GFP recombinant virus on Marc145 cells is shown in FIG. 4.
(9) Off-target detection of transfected cells
Comparing the selected sgRNA sequence with a Marc145 cell genome, searching for a corresponding highly repetitive sequence, selecting gene loci CPEB2-AS1, F11-AS1, MAGI1 and NUP210 which may have off-target, designing a corresponding Q-PCR detection primer, extracting mRNA from a cell sample transfected for 48 hours, inverting to obtain a cDNA sample, and performing Q-PCR to detect the relative expression amount of the gene mRNA which may have off-target effect, thereby judging whether the off-target condition may exist.
The results show that the NES-Cas13D system mediates the PRRSV-GFP virus to generate efficient knockdown, the cell activity is not obviously changed, and no off-target condition occurs (figure 5, A-D).