CN111440801A - sgRNA for targeted knockout of human NKG2A/K L RC1 gene, expression vector, kit and application thereof - Google Patents

Abstract

The invention relates to the field of genetic engineering, discloses sgRNA for targeted knockout of human NKG2A/K L RC1 gene, and the sgRNA has any nucleotide sequence of SEQ ID NO.1-56, and also discloses an sgRNA composition, an expression vector, a CRISPR/Cas9 system, a kit and application thereof in preparation of immune cell medicaments for targeted knockout of human NKG2A/K L RC1 gene, wherein the specific targeted human NKG2A/K L RC1 gene prepared by the invention can accurately target human NKG2A/K L RC1 gene and realize effective knockout, and meanwhile, the obtained human NKG2A/K L RC1 gene knockout immune cells are suitable for various tumor cell models, and the preparation method has the advantages of simple steps, good specificity of sgRNA and high knockout efficiency of the CRISPR/Cas9 targeted system.

Description

sgRNA for targeted knockout of human NKG2A/K L RC1 gene, expression vector, kit and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to sgRNA, an expression vector, a kit and application thereof, wherein the sgRNA is used for targeted knockout of a human NKG2A/K L RC1 gene.

Background

The CRISPR/Cas system was developed from the adaptive immune system of bacteria and archaea against foreign viruses or plasmids, and the CRISPR sequence and its related gene (Cas gene) act together and are of great interest to the biological world due to their characteristic RNA-mediated endonuclease activity. Among them, the type II endonuclease Cas9, which is derived from Streptococcus Pyogenes as a representative, is most widely used because it has only one subunit and has the simplest structure. The CRISPR/Cas9 system locates a target gene through a small-guide RNA (sgRNA) sequence for recognizing a specific sequence, guides Cas9 to cut the target sequence, causes Double-strand breaks (DSB), and generates insertion or deletion of a base in a Non-Homologous recombination (NHEJ) DNA repair mode under the condition without a template, thereby causing frameshift mutation to achieve the purpose of gene knockout.

Compared with ZFN (Finger nucleus) and TA L EN (Transcription Activator-like effector nuclei), which require researchers to design and plant a pair of specific nucleases according to target genes, the CRISPR/Cas9 technology can quickly and efficiently target any one or more genes, and has the advantages of simple operation, high-throughput preparation, low cost and the like.

In the CRISPR/Cas9 system, targeted cleavage of Cas9 is achieved by virtue of sgRNA recognizing the target sequence. Although the target sequence has only 20 nucleotides, the design is very simple, but the probability of non-specific binding (off-target) is relatively large. Off-target can cause gene mutation other than the target gene, and bring unpredictable influence to human body. The off-target efficiency is a considerable potential safety hazard for clinical application of gene therapy and the like, and is also a main reason for limiting the development and application of the technology. Therefore, the specificity and targeting accuracy of sgrnas for a gene of interest are prerequisites for specific knockdown of the gene of interest. Therefore, the design and preparation of sgrnas with high specificity and accuracy are key to CRISPR/Cas9 gene knockout.

In clinical tumor therapy, immunotherapy using antibodies that block Immune checkpoint on the surface of Immune cells is a common treatment approach, Immune Checkpoints (Immune Checkpoints) are signal pathways that regulate systemic Immune homeostasis and tolerance, numerous studies have shown that tumor cells express high levels of Immune checkpoint ligands on the surface, and that tumor immunity-inhibiting effects are achieved by binding Immune Checkpoints of Immune cells, this effect is particularly prominent in tumor suppression by tumor antigen-specific effector T cells and natural killer cells (NK), blocking of specific Immune Checkpoints, such as PD-1, can enhance anti-tumor Immune responses, recent studies published in international Cell, have shown that blocking of Immune checkpoint NKG2A with antibodies can promote tumor killing of NK cells and effector T cells, and that NKG2A has the most significant clinical value in clinical trials following CT L a-4, PD-1/L1, and IDO 2.

However, treatment with immune checkpoint antibodies is not effective for all cancers. The PD-1 antibody has a good therapeutic effect on melanoma, but has an effect only on a part of kidney cancer and lung cancer. Other immune checkpoint antibody drugs are also in clinical trials, such as NKG2A, and many tumor therapies using NKG2A antibodies have been reported. However, there are many disadvantages such as short-term effect of antibody drugs, diversity of immune check points, long drug development time, and high price of antibodies.

In contrast, the advantages of CRISPR/Cas9 technology are self evident. Immune cells are modified by using a CRISPR/Cas9 technology, and genes related to surface immune checkpoints are knocked out, so that a new strategy is provided for realizing tumor immunotherapy.

In conclusion, sgrnas targeting immune checkpoint genes with accuracy and specificity for various cells are designed and prepared, and the improvement of the gene editing rate is the key point of CRISPR/Cas9 specific gene knockout.

Disclosure of Invention

One of the purposes of the invention is to provide a sgRNA for targeted knockout of human NKG2A/K L RC1 gene, so as to overcome the defects of short effect of antibody drug action, diversity of immune check points, long drug development time, high antibody price and low gene editing efficiency of CRISPR/Cas9 technology in the prior art.

The technical scheme is that the sgRNA for targeted knockout of the human NKG2A/K L RC1 gene has the nucleotide sequence shown in any one of SEQ ID NO. 1-56.

Further, the method for designing the nucleotide sequence of the sgRNA comprises the following steps:

s1, selecting common exons of different splicing forms on an NKG2A/K L RC1 gene;

s2, finding out the sequence of 5 '-GGN (19) GG-3', 5 '-GN (20) GG-3' or 5 '-N (21) GG-3' in the common exon after the first initiation codon ATG.

Further, the sequence does not comprise 3 or more consecutive bases a or T.

Furthermore, the site of the sequence is positioned in the first half section of the whole gene and cannot be too close to the initiation codon ATG, so that the formation of a truncated gene caused by the initiation of another downstream ATG after transcription is prevented, and the effect of ensuring complete inactivation of the gene is achieved.

Further, the sequence is a single sequence or a paired sequence, and the distance between the paired sequences is 0-200 bp.

The invention also aims to provide an expression vector for targeted knockout of a human NKG2A/K L RC1 gene, and the technical scheme is that the expression vector expresses the sgRNA.

The third purpose of the invention is to provide a CRISPR/Cas9 system for targeted knockout of human NKG2A/K L RC1 gene, and the technical scheme is that the CRISPR/Cas9 system comprises the sgRNA and Cas9 protein expression plasmid.

The fourth purpose of the invention is to provide a kit for targeted knockout of human NKG2A/K L RC1 gene, and the technical scheme is that the kit comprises a sgRNA vector and a Cas9 protein.

The fifth purpose of the invention is to provide a sgRNA composition for targeted knockout of a human NKG2A/K L RC1 gene, and the technical scheme is that the sgRNA composition comprises at least two sgRNAs with nucleotide sequences of SEQ ID NO. 1-56.

The sixth purpose of the present invention is to provide an application of the sgRNA in preparing an immune cell drug for treating tumor diseases.

The seventh object of the present invention is to provide a use of the sgRNA composition in preparing an immune cell drug for treating a tumor disease.

The invention is realized by the following method:

1. design and selection of sgRNA sequences targeting the NKG2A/K L RC1 gene:

(a) finding the sequence of the NKG2A/K L RC1 gene through NCBI database;

(b) selecting different splicing forms of common exons on the NKG2A/K L RC1 gene;

(c) finding the sequence of 5 '-GGN (19) GG-3', 5 '-GN (20) GG-3' or 5 '-N (21) GG-3' in the consensus exon after the first initiation codon ATG; wherein, the sequence is a single sequence or a paired sequence, and the interval distance of the paired sequence is 0-200 bp;

(d) selecting a sequence of which the site is positioned in the first half of the whole gene and can not be too close to the initiation codon ATG, and simultaneously, the sequence does not contain more than 3 continuous bases A or T;

(e) determining whether the target sequence of the sgRNA is unique using B L AST of NCBI or B L AT of UCSC;

2. construction of oligonucleotide double strands of sgRNA:

(a) adding appropriate restriction enzyme cohesive ends 5' to the selected sgRNA sequence, adding ACCG to the sgRNA sequence attached to the Bsa I cleavage site, adding CACC to the sgRNA sequence attached to the Bbs I cleavage site, resulting in a Forward oligonucleotide (Forward oligo);

(if the sgRNA sequence does not start with a G at the 5 'end, then add one G at the 5' end followed by the addition of the endonuclease sticky end);

(b) obtaining a complementary strand of the corresponding DNA according to the selected sgRNA sequence, and adding AAAC at the 5' end of the complementary strand to obtain a Reverse oligonucleotide (Reverse oligo);

(c) synthesizing the forward oligonucleotide and the reverse oligonucleotide, respectively;

(d) carrying out denaturation and annealing on the synthesized forward and reverse sgRNA oligonucleotides in pairs, and enabling the sgRNA oligonucleotides to form a double-stranded structure which can be accessed into a linear U6 eukaryotic expression pG L3-2U 6-sgRNA body through base pairing;

the double-stranded structure is as follows:

construction of sgRNA oligonucleotide plasmids:

(a) a linearized pG L3-2U 6-sgRNA vector (structure is shown in figure 1, and sequence is shown in SEQ ID NO. 77);

(b) connecting the annealed sgRNA oligonucleotide double strand with a linearized pG L3-2U 6-sgRNA vector to obtain pG L3-2U 6-NKG2A/K L RC1-sg plasmid;

(c) amp + plates (100. mu.g/m L) were transformed and coated;

(d) picking single clone, shaking the strain in L B culture solution at 37 ℃ overnight;

(e) taking part of the bacterial liquid, and detecting whether the bacterial liquid is correctly inserted into the carrier by using a sequencing specific primer of SEQ ID NO. 71; extracting the Plasmid from the residual bacterial liquid by using an EndoFree Plasmid kit (QIAGEN);

4. the NKG2A/K L RC1 gene knockout immune cells are obtained by transfecting immune cells:

the plasmid carrying the sgRNA oligonucleotide was mixed with the Cas9 plasmid (structure shown in FIG. 2) having the sequence of SEQ ID NO.76, according to the manual of Human T Cell Nucleofector Kit (L naza), and cells were co-transfected.

5. The NKG2A/K L RC1 gene was knocked out by flow cytometry.

Furthermore, a pair of sgRNAs adjacent to each other (targeting initiation sites on human NKG2A/K L RC1 genes and spaced at a distance of 0-200bp) can be utilized to remarkably improve the knockout efficiency, and specifically, after the design, selection and synthesis of a sgRNA oligonucleotide targeting human NKG2A/K L RC1 genes, a pair of sgRNA oligonucleotides targeting human NKG2A/K L RC1 genes are connected with linearized pG L3-2U 6-sgRNA vectors to obtain vectors pG L3-2U 6-NKG2A/K L RC 1-sgG 1-sgRNA 2 plasmids containing a pair of sgRNA oligonucleotides targeting human NKG2A/K L RC1 genes, and then cell transfection is carried out.

The invention designs and synthesizes a group of sgRNAs specifically targeting the gene in the forward direction and the reverse direction by specifically knocking out human NKG2A/K L RC1 genes in cells by using a CRISPR/Cas9 technology, and the sgRNAs are connected with a linear pG L3-2U 6-sgRNA vector which is modified to load 2 sgRNAs to form a plasmid, and the knockout of the human NKG2A/K L RC1 genes can be realized by successfully transfecting the cells with the vector and the Cas9 plasmid at the same time.

The invention provides a strategy for specifically knocking out human NKG2A/K L RC1 genes quickly, efficiently and at low cost by using a CRISPR/Cas9 technology in combination with sgRNA of a specific targeting human NKG2A/K L RC1 gene, effectively solves the problems existing in the existing NKG2A antibody treatment, realizes the effect of permanently inhibiting an immune checkpoint NKG2A, can knock out a plurality of coding sequences of the human NKG2A/K L RC1 gene simultaneously and can knock out a plurality of target genes simultaneously, the efficient sgRNA prepared by the method can be produced in large scale only by small-scale synthesis, and meanwhile, immune cells for knocking out the human NKG2A/K L RC1 gene have great application prospects in the field of tumor cell treatment.

Compared with the prior art, the invention has the advantages that:

1. the present invention can directly edit various cells such as T cells, NK cells, B cells, DC cells, granulocytes, eosinophils, basophils, and the like contained in PBMCs by electrotransfection.

2. According to the invention, by limiting the screening conditions of the sgRNA sequences and combining a pair of sgRNA compositions to knock out genes, the off-target efficiency is greatly reduced, the gene editing efficiency and the feasibility of clinical use are improved, and the safety risk is reduced;

3. compared with the temporary blocking effect of the NKG2A antibody in the prior art, the direct knockout of the NKG2A/K L RC1 gene can realize permanent effect;

4. the antibody drug can not simultaneously block a plurality of inhibitory receptors at present, and the invention can knock out a plurality of coding sequences of NKG2A and a plurality of target genes;

5. the number of the existing effective NKG2A antibodies is very small, and the research and development difficulty is high, so that the invention provides a group of efficient sgRNAs aiming at human NKG2A/K L RC1 genes;

6. the antibody drug action can only aim at extracellular targets, and the invention can aim at both extracellular targets and intracellular targets;

7. compared with the time-consuming, labor-consuming and expensive development of antibody drugs, the sgRNA is used for synthesizing polynucleotide fragments in small quantity, so that the large-scale production can be realized.

Drawings

FIG. 1 is a diagram of the construction of pG L3-2U 6-sgRNA vector;

figure 2 is a structural diagram of the Cas9 plasmid;

FIG. 3 is a graph showing the results of flow cytometry.

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the following.

As used herein, the term "consensus exons of different splicing forms" refers to exons that are shared by multiple gene sequences that are variably spliced; whereas variable splicing refers to the process by which exons of RNA produced by transcription of major genes or mRNA precursors are reconnected by RNA splicing in a variety of ways.

As used herein, the term "expression vector" refers to a vector in which an expression element (e.g., promoter, RBS, terminator, etc.) is added to the basic backbone of a cloning vector to enable the expression of a gene of interest.

As used herein, the term "vector" refers to an expression vector capable of expressing a target protein in an appropriate host cell and a genetic construct comprising essential regulatory elements to which a gene insert is operably linked in an expressible manner.

As used herein, the term "Cas 9 protein" refers to the major protein element of the CRISPR/Cas9 system that forms a complex with crRNA (crisprrna) and tracrRNA (transactivating crRNA) to form an activated endonuclease or nickase.

Furthermore, Cas9 proteins may include not only wild-type Cas9, but also inactivated Cas9(dCas9) or Cas9 variants such as Cas9 nickase. Wherein the inactivated Cas9 may be RFN comprising a fokl nuclease domain that binds to dCas9 or dCas9 that binds to a transcription activator or repressor domain; the Cas9 nickase may be D10ACas9 or H840ACas9, but is not limited thereto.

For example, the Cas9 protein may be derived from Streptococcus pyogenes (Streptococcus pyogenes), Francisella novarus (Francisella novicida), Streptococcus thermophilus (Streptococcus thermophilus), Legionella pneumophila (L EGIONella pneumoniae), Listeria innocua (L isteria innocula), or Streptococcus mutans (Streptococcus mutans).

Example 1

Design and synthesis of sgRNA for specifically targeting human NKG2A/K L RC1 gene in human NKG2A/K L RC1 gene by CRISPR/Cas9 specific knockout.

1. Design of sgRNA targeting human NKG2A/K L RC1 gene:

(a) selecting a sequence of 5 '-GGN (19) GG-3', 5 '-GN (20) GG-3' or 5 '-N (21) GG-3' on the human NKG2A/K L RC1 gene;

(b) selecting a sequence of a targeting site positioned on an exon of a human NKG2A/K L RC1 gene to ensure that the gene is easy to completely inactivate;

(c) the sgRNA targets the human NKG2A/K L RC1 gene at the position of the common exon in different splicing forms;

(d) determining whether the target sequence of the sgRNA is unique using B L AST of NCBI or B L AT of UCSC;

according to the method, 56 sgRNAs targeting human NKG2A/K L RC1 genes are designed in total, and the sequences are respectively shown in sequence tables SEQ ID NO. 1-56;

2. selection of sgRNA targeting the human NKG2A/K L RC1 gene:

(a) the sgRNA sequence is not spaced too close to the first ATG initiation codon;

(b) the targeting site of sgRNA on human NKG2A/K L RC1 gene is located in front of the whole gene;

(c) the sgRNA sequence does not comprise 3 or more consecutive bases a or T;

(d) selecting paired sequences separated by 0-200bp on human NKG2A/K L RC1 gene;

according to the method, 23 sequences (shown as sequences SEQ ID NO.2, 20-27, 29, 32-33, 36-40, 42, 46-47 and 51-53) are matched in 56 targeting sgRNAs (shown as sequences SEQ ID NO. 1-56);

for experimental verification, 5 sequences (shown as SEQ ID NO.2, 20, 21, 23 and 26 respectively) meeting the screening conditions and 2 sequences (shown as SEQ ID NO.6 and 8 respectively) not meeting the screening conditions are selected from the sequences for subsequent experiments;

3. synthesis and construction of sgRNA oligonucleotides targeting the human NKG2A/K L RC1 gene:

adding ACCG or CACC to the 5 ' end of 7 sgRNA sequences (shown as sequences SEQ ID NO.2, 6, 8, 20, 21, 23 and 26 respectively) selected according to the previous steps to obtain Forward oligonucleotide (Forward oligo), and adding ACCG or CACC after adding a G to the 5 ' end if the sequence itself does not start with a G at the 5 ' end; obtaining a complementary strand according to the selected sgRNA, and adding AAAC at the 5' end to obtain a Reverse oligonucleotide (Reverse oligo); the oligonucleotides are respectively synthesized, and the Forward oligo and Reverse oligo of the synthesized sgRNA oligonucleotide are denatured in pairs and annealed to form double-stranded sgRNA with sticky ends, and the mode is as follows:

the denaturation and annealing system comprises:

5μl Forward oligo(100μM)

5μl Reverse oligo(100μM)

run in a PCR instrument according to the following touch down program: at 95 ℃ for 2 min; 95-25 deg.C at-1 deg.C/min; keeping at 4 ℃;

the sequences of the obtained forward oligonucleotide and reverse oligonucleotide of SEQ ID NO.2 are respectively shown as SEQ ID NO.57 and 58;

the obtained forward oligonucleotide and reverse oligonucleotide sequences of SEQ ID NO.6 are shown as SEQ ID NO.59 and 60 respectively;

the obtained forward oligonucleotide and reverse oligonucleotide sequences of SEQ ID NO.8 are respectively shown as SEQ ID NO.61 and 62;

the obtained forward oligonucleotide and reverse oligonucleotide sequences of SEQ ID NO.20 are respectively shown as SEQ ID NO.63 and 64;

the obtained forward oligonucleotide and reverse oligonucleotide sequences of SEQ ID NO.21 are shown in SEQ ID NO.65 and 66 respectively;

the obtained forward oligonucleotide and reverse oligonucleotide sequences of SEQ ID NO.23 are shown as SEQ ID NO.67 and 68 respectively;

the obtained forward oligonucleotide and reverse oligonucleotide sequences of SEQ ID NO.26 are shown as SEQ ID NO.69 and 70 respectively.

Example 2

Human NKG2A/K L RC1 genes (a pair of sgRNAs used for targeting human NKG2A/K L RC1 genes in the embodiment are respectively shown as sequences in SEQ ID NO.2, 6, 8, 20, 21, 23 and 26) are knocked out by using CRISPR/Cas 9.

The method comprises the following steps:

1. pG L3-2U 6-sgRNA vector with linearized sequence shown as sequence SEQ ID NO.77

The enzyme digestion system and conditions were as follows:

2μg pGL3-2U6-sgRNA

5μL CutSmart Buffer

1μL BsaI(NEB)

adding water to 50 mu L37 ℃, and reacting for 1 hour;

after the enzyme digestion is finished, carrying out 1% agarose gel electrophoresis, and recovering pG L3-2U 6-sgRNA vector by adopting an EndoFree plasma Kits (QIAGEN);

2. double-stranded sgRNA (2), sgRNA (8), sgRNA (21) and sgRNA (23) (shown as sequences SEQ ID No.2, 8, 21 and 23 respectively) oligonucleotides which are obtained after denaturation and annealing and can be connected into a U6 eukaryotic expression vector are connected with a linearized pG L-2U 6-sgRNA vector to obtain pG L3-2U 6-NKG2A/K L RC1-sg (2), pG L-2U 6-NKG2A/K L RC1-sg (8), pG L-2U 6-NKG2A/K L RC1-sg (21) and pG L-2U 6-NKG2A/K L RC1-sg (23) plasmids;

the linking system is as follows:

mu. L50 mu.M annealing product (double-stranded sgRNA oligonucleotides, forward oligonucleotides are shown in SEQ ID No.57, 61, 65, 67, and reverse oligonucleotides are shown in SEQ ID No.58, 62, 66, 68)

1 μ L linearized pG L3-2U 6-sgRNA vector (25 ng/. mu. L)

1μL T4 ligation Buffer

0.5μLT4 ligase(NEB)

4.5 mu L sterilized water

Incubating at 16 ℃ for 2 hours;

3. the ligation product obtained in the above step was transformed into 100. mu. L DH5 α competent cells (TransGen Biotech) and plated with Amp + plates (100. mu.g/m L), and single clones were selected for shake culture in L B medium at 37 ℃ for 6 h;

4. adding part of the shake culture liquid into L B culture medium, shaking for overnight culture, and extracting plasmid with EndoFreePlamidskit (QIAGEN) to obtain pG L3-2U 6-NKG2A/K L RC1-sg (2), pG L3-2U 6-NKG2A/K L RC1-sg (8), pG L3-2U 6-NKG2A/K L RC1-sg (21), and pG L3-2U 6-NKG2A/K L RC1-sg (23);

5. linearized pG L3-2U 6-NKG2A/K L RC1-sg (2), pG L3-2U 6-NKG2A/K L RC1-sg (8), pG L3-2U 6-NKG2A/K L RC1-sg (21), pG L3-2U 6-NKG2A/K L RC1-sg (23);

2 μ g plasmid

5μL CutSmart Buffer

1μLBbsI(NEB)

Adding water to 50 mu L37 ℃, and reacting for 1 hour;

after the completion of the digestion, 1% agarose gel electrophoresis was performed, and plasmids pG L3-2U 6-NKG2A/K L RC1-sg (2), pG L3-2U 6-NKG2A/K L RC1-sg (8), pG L3-2U 6-NKG2A/K L RC1-sg (21), pG L3-2U 6-NKG2A/K L RC1-sg (23) were recovered using EndoFree plasma Kits (QIAGEN);

6. the double-stranded sgRNA (6), sgRNA (20) and sgRNA (26) (shown in sequences SEQ ID NO.6, 20 and 26 respectively) which are obtained after denaturation and annealing and can be connected into a U eukaryotic expression vector are connected with linearized pG 3-2U-NKG 2/K RC-sg (2), pG 03-2U-NKG 2/K1 RC-sg (8), pG 23-2U-NKG 2/K3 RC-sg (21), pG 43-2U-NKG 2/K5 RC-sg (23) to obtain pG 3-2U-NKG 2/K RC-sg (2) -sg (6), pG 3-2U-NKG 2/K RC-sg (8) -sg (6), pG 3-2U-NKG 2/K RC-sg (21) -sg (26) and pG 3-2U-NKG 2/K-sg (23) -sg (20) plasmids;

the linking system is as follows:

mu. L50 mu.M annealing product (double-stranded sgRNA oligonucleotides, wherein the forward oligonucleotides are shown in SEQ ID NO.59, 63 and 69 of the sequence table, and the reverse oligonucleotides are shown in SEQ ID NO.60, 64 and 70 of the sequence table)

1 μ L linearized plasmid (25 ng/. mu.l)

1μL T4 ligation Buffer

0.5μLT4 ligase(NEB)

4.5 mu L sterilized water

Incubating at 16 ℃ for 2 hours;

7. the ligation product obtained in the above step was transformed into 100. mu.l DH5 α competent cells (TransGen Biotech) and plated on Amp + plates (100. mu.g/m L), and single clones were selected for shake culture in L B medium at 37 ℃ for 6 h;

8. adding part of the shake culture liquid into L B culture medium, shaking for overnight culture, and extracting plasmid with EndoFreePlamidskit (QIAGEN) to obtain plasmid pG L3-2U 6-NKG2A/K L RC1-sg (2) -sg (6), pG L3-2U 6-NKG2A/K L RC1-sg (8) -sg (6), pG L3-2U 6-NKG2A/K L RC1-sg (21) -sg (26), and pG L3-2U 6-NKG2A/K L RC1-sg (23) -sg (20);

9. cell culture and transfection

(a) Inoculating human immunocyte cells in RPMI-1640 culture medium containing 10% autologous serum, penicillin (100U/m L) and streptomycin (100 μ g/m L);

(b) dividing the cells into 6-well plates before transfection, performing transfection when the density is 70-80%, and collecting the cells by centrifugation (the relative centrifugal force is 90g, and the time is 10 min);

(c) mixing 5 μ g of pG L3-2U 6-NKG2A/K L RC1-sg (2) -sg (6), pG L3-2U 6-NKG2A/K L RC1-sg (8) -sg (6), pG L3-2U 6-NKG2A/K L RC1-sg (21) -sg (26), pG L3-2U 6-NKG2A/K L RC1-sg (23) -sg (20) with 10 μ g of Cas9 plasmid according to the manual of Human T Cell Nuclear effector Kit (L naza), performing electrotransfection, placing the mixture in an incubator at 37 ℃ for 10min, transferring the cells to a culture flask, changing the culture solution after 6-8 hours, and collecting the cells after 48 hours;

10. flow cytometry detection

(a) Take 1 × 10⁶The transfected and cultured cells are washed twice by PBS in a centrifugal mode;

(b) after resuspending the cells with 100 μ L PBS, BSA was added and incubated for 5 min at room temperature;

(c) adding PE-anti-human NKG2A antibody, and incubating for 30 minutes at 4 ℃ in the dark;

(d) washing the cells once with PBS;

(e) the expression of cell-surface NKG2A was detected by flow cytometry, and the results are shown in fig. 3.

Example 3

For experimental verification, 4 sequences (shown as SEQ ID NO.20, 22, 26, and 27, respectively) were selected from the 23 sequences selected in step 2 of example 1 again, and pG L3-2U 6-NKG2A/K L RC1-sg (22) -sg (20), pG L3-2U 6-NKG2A/K L RC1-sg (26) -sg (20), pG L3-2U 6-NKG2A/K L RC1-sg (26) -sg (27) were prepared in the same manner as in step 3 of example 1 and steps 1 to 8 of example 2, and cultured cells were obtained in the same manner as in step 9 of example 2, and finally examined in the same manner as in step 10 of example 2.

As shown in FIG. 3, the expression reduction rates (i.e., knockout rates) of cell surface NKG 2L 03 were 25.4% and 14.3% after the plasmids pG L3-2U 6-NKG2A/K L RC1-sg (6) and pG L-2U L-NKG 2/K L RC 72-sg (6), respectively, while the expression reduction rates (i.e., knockout rates) of cell surface NKG 2L were respectively 25.4% and 14.3% after the plasmids were treated with pG 8472-2U 6-sG (2) -sG 72-K L/K L RC 3-sG (21) -sG 72-sG (26), pG L-2U L-NKG 2-L-sG (23) -sG 72-sG (22) -sG (20), pG 3663-2U L-sG 2-L-sG (72) and the expression rates of pG L-sG 72-sG (72) were respectively, as shown by pairs of the continuous expression rates of pG L-sG 3-sG (72-sG) and the plasmids were respectively, as shown by the continuous expression rates of the expression rates (19-L-sG (23-L), the sequences) and the continuous expression rates after the plasmids were respectively, the plasmids were more than those of the continuous expression rates (23-L-72-19-L-19-L-19-sG-L-19, the sequences were respectively, the sequences were shown by the sequences, the sequences were more than the sequences, the sequences were shown by the sequences were more than.

Note: since the Peripheral Blood Mononuclear Cells (PBMCs) are edited in the present invention, and include a plurality of cells such as T cells, NK cells, B cells, DC cells, granulocytes, eosinophils, and basophils, and the editing rate differs for each cell, the editing rate obtained for each sample in fig. 3 is the average value of the various types of cells included in the sample.

The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

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<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ttcctgttca gttgctaaaa 20

<210>9

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

atctgccccc aaacccaaag 20

<210>10

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gttttcgttg ctgcctcttt 20

<210>11

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

aagcttctca ggattttcaa 20

<210>12

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ttgctgcctc tttgggtttg 20

<210>13

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

ttgggtttgg gggcagattc 20

<210>14

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

aatcctgaga agctttttga 20

<210>15

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

gttgctgcct ctttgggttt 20

<210>16

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

tgctgcctct ttgggtttgg 20

<210>17

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

aaagcttctc aggattttca 20

<210>18

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

aaaccttcaa aaagcttctc 20

<210>19

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

atggagcttt tattgccttt 20

<210>20

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

aacaactatc gttaccacag 20

<210>21

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>21

gctccagaga agctcattgt 20

<210>22

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>22

gaagctcatt gttgggatcc 20

<210>23

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>23

aagctcattg ttgggatcct 20

<210>24

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>24

atcccaacaa tgagcttctc 20

<210>25

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>25

ctccagagaa gctcattgtt 20

<210>26

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>26

agataagaca gataattccc 20

<210>27

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>27

atgagcttct ctggagctga 20

<210>28

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>28

aattatctgt cttatcttaa 20

<210>29

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>29

tcttatctta atggcctctg 20

<210>30

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>30

tttctgagtt cttgtattca 20

<210>31

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>31

ctttctgagt tcttgtattc 20

<210>32

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>32

actggagttc ttcgaagtac 20

<210>33

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>33

tccaacagtt gttactacat 20

<210>34

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>34

attatctata gaaagcagac 20

<210>35

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>35

aacttgggaa gagagtttgc 20

<210>36

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>36

cagttgttac tacattggta 20

<210>37

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>37

taatccactc ctcaggacaa 20

<210>38

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>38

tcattgtggc cattgtcctg 20

<210>39

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>39

gtggccattg tcctgaggag 20

<210>40

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>40

accaatgtag taacaactgt 20

<210>41

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>41

tggtaaggaa agaagaactt 20

<210>42

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>42

gaatatgtaa tccactcctc 20

<210>43

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>43

ttggtaagga aagaagaact 20

<210>44

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>44

ccatcatttc accatcctca 20

<210>45

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>45

ccatgaggat ggtgaaatga 20

<210>46

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>46

catccatggg tgacaatgaa 20

<210>47

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>47

atgggtgaca atgaatggtt 20

<210>48

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>48

atttcaccat cctcatggat 20

<210>49

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>49

aaaccattca ttgtcaccca 20

<210>50

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>50

acgaaacaca ccaatccatg 20

<210>51

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>51

gtaacagcag tcatcatcca 20

<210>52

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>52

taacagcagt catcatccat 20

<210>53

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>53

aacacaccaa tccatgagga 20

<210>54

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>54

tattattgaa gatccacact 20

<210>55

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>55

cgacttaaat cagcccagtg 20

<210>56

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>56

atattattga agatccacac 20

<210>57

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>57

accgggtctg agtagattac tcct 24

<210>58

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>58

aaacaggagt aatctactca gacc 24

<210>59

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>59

caccgaggca gcaacgaaaa cctaa 25

<210>60

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>60

aaacttaggt tttcgttgct gcctc 25

<210>61

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>61

accggttcct gttcagttgc taaaa 25

<210>62

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>62

aaacttttag caactgaaca ggaac 25

<210>63

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>63

caccgaacaa ctatcgttac cacag 25

<210>64

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>64

aaacctgtgg taacgatagt tgttc 25

<210>65

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>65

accggctcca gagaagctca ttgt 24

<210>66

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>66

aaacacaatg agcttctctg gagc 24

<210>67

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>67

accggaagct cattgttggg atcct 25

<210>68

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>68

aaacaggatc ccaacaatga gcttc 25

<210>69

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>69

caccgagata agacagataa ttccc 25

<210>70

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>70

aaacgggaat tatctgtctt atctc 25

<210>71

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>71

tgaacggatc tcgacggt 18

<210>72

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>72

ataagaaacg tgtttaggct tg 22

<210>73

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>73

aaccctcatc tcccattgta 20

<210>74

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>74

ggaaagagaa gggagtgctc 20

<210>75

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>75

tacgattgca tcagaagcg 19

<210>76

<211>8113

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>76

tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 60

gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc 120

ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc 180

tcggcatgga cgagctgtac aagtaagttt aaacccgctg atcagcctcg actgtgcctt 240

ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 300

ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 360

gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 420

atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 480

ggggctctag ggggtatccc cattgcgttg cgctcactgc ccgctttcca gtcgggaaac 540

ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 600

gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 660

gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 720

ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 780

ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 840

cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 900

ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 960

tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 1020

gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 1080

tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 1140

gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 1200

tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 1260

ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 1320

agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 1380

gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 1440

attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 1500

agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 1560

atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 1620

cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 1680

ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 1740

agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 1800

tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 1860

gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 1920

caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 1980

ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 2040

gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 2100

tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 2160

tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 2220

cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 2280

cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 2340

gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 2400

atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 2460

agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 2520

ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca 2580

ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg 2640

tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga 2700

ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac 2760

gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg 2820

ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc 2880

cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca 2940

tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg 3000

cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg 3060

acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt 3120

ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca 3180

tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg 3240

tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact 3300

ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag 3360

ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact 3420

atagggagac ccaagctggc tagcaccatg gacaagaaat actctattgg actggatatc 3480

gggacaaact ccgttggctg ggccgtcata accgacgagt ataaggtgcc aagcaagaaa 3540

ttcaaggtgc tgggtaatac tgaccgccat tcaatcaaga agaacctgat cggagcactc 3600

ctcttcgact ccggtgaaac cgctgaagct actcggctga agcggaccgc aaggcggaga 3660

tacacccgcc gcaagaatcg gatatgttat ctgcaagaga tctttagcaa cgaaatggct 3720

aaggtggacg actccttctt tcaccgcctg gaagagagct ttctggtgga ggaggataag 3780

aaacacgaga ggcaccctat attcggaaat atcgtggatg aggtggctta ccatgaaaag 3840

tatcctacaa tctaccatct gaggaagaag ctggtggaca gcaccgataa agcagacctg 3900

aggctcatct atctggccct ggctcatatg ataaagttta gaggacactt tctgatcgag 3960

ggcgacctga atcccgataa ttccgatgtg gataaactct tcattcaact ggtgcagaca 4020

tataaccaac tgttcgagga gaatcccata aacgcttctg gtgtggatgc caaggctatt 4080

ctgtccgctc ggctgtccaa gtcacgcaga ctggagaatc tgattgccca actgccagga 4140

gaaaagaaga acggcctgtt tgggaacctc atcgccctga gcctgggcct gacacctaac 4200

ttcaagtcca attttgatct ggccgaagat gctaaactcc agctctccaa ggacacctat 4260

gacgatgatc tggacaacct gctcgcacag ataggcgacc agtacgccga tctctttctg 4320

gctgctaaga atctctccga cgccattctg ctgagcgaca tactccgggt caacactgag 4380

atcaccaaag cacctctgag cgcctccatg ataaaacgct atgatgaaca ccatcaagac 4440

ctgactctgc tcaaagccct cgtgaggcaa cagctgccag agaagtacaa agagatattc 4500

ttcgaccaga gcaagaatgg atatgccgga tacatcgatg gcggagcatc acaggaagaa 4560

ttttacaagt tcatcaaacc aatcctcgag aagatggacg gtactgaaga gctgctggtg 4620

aagctgaaca gggaggacct gctgaggaag cagaggacct ttgataatgg ctccattcca 4680

catcagatac acctgggaga gctgcatgca atcctccgca ggcaggagga tttctatcct 4740

ttcctgaagg ataaccggga gaagatagag aagatcctga ccttcaggat cccttattac 4800

gtcggccctc tggctagagg caactcccgc ttcgcttgga tgaccaggaa atctgaggag 4860

acaattactc cttggaactt cgaagaggtc gtggataagg gcgcaagcgc ccagtcattc 4920

atcgaacgga tgaccaattt cgataagaac ctgcccaacg agaaggtcct gcccaaacat 4980

tcactcctgt acgagtattt caccgtctat aacgagctga ctaaagtgaa gtacgtgacc 5040

gagggcatga ggaagcctgc cttcctgtcc ggagagcaga agaaggctat cgttgatctg 5100

ctcttcaaga ctaatagaaa ggtgacagtg aagcagctca aggaggatta ctttaagaag 5160

atcgaatgct ttgactcagt ggaaatctct ggcgtggagg accgctttaa tgccagcctg 5220

ggcacttacc atgatctgct gaagataatc aaagacaaag atttcctcga taatgaggag 5280

aacgaggaca tcctggaaga tatcgtgctg accctgactc tgttcgagga tagagagatg 5340

atcgaagagc gcctgaagac ctatgcccat ctgtttgacg ataaagtcat gaaacagctc 5400

aagcggcggc gctacactgg gtggggtaga ctctccagga aactcataaa cggcatccgc 5460

gacaaacaga gcggaaagac catcctggat ttcctgaaat ccgacggatt cgctaacagg 5520

aacttcatgc aactgattca cgatgactct ctgacattta aagaggacat ccagaaggca 5580

caggtgagcg gtcaaggcga cagcctgcac gagcacatcg ccaacctcgc tggatcaccc 5640

gccataaaga agggaatact gcagacagtc aaggtcgtgg acgaactcgt caaagtgatg 5700

ggtcggcaca agccagagaa tatcgttatc gaaatggcaa gggagaacca aaccacccag 5760

aagggccaga agaactctcg ggaacggatg aaaagaatcg aagagggaat taaggagctg 5820

ggatctcaga tactgaagga gcaccctgtg gagaatacac agctccagaa cgagaaactc 5880

tacctgtact acctccagaa cgggcgggac atgtacgttg accaggaact cgacatcaac 5940

cggctgtccg attatgacgt ggaccatatt gttccacagt ccttcctcaa agatgactcc 6000

attgacaaca aggtgctgac cagatccgat aagaatcgcg gtaagtctga caatgttcca 6060

tcagaagagg tggtcaagaa gatgaagaat tactggcggc agctcctcaa cgccaaactg 6120

atcacccagc ggaagtttga caatctgact aaggcagaaa gaggaggtct gagcgaactc 6180

gacaaggccg gctttattaa gaggcaactg gtcgaaacac gccagattac caaacacgtg 6240

gcacaaatcc tcgactctag gatgaacact aagtacgatg agaacgataa gctgatcagg 6300

gaagtgaaag tgataactct gaagagcaag ctggtgtctg acttccggaa ggactttcaa 6360

ttctacaaag ttcgcgaaat aaacaattac catcatgctc acgatgccta tctcaatgct 6420

gtcgttggca ccgccctgat caagaaatac cctaaactgg agtctgagtt cgtgtacggt 6480

gactataaag tctacgatgt gaggaagatg atagcaaagt ctgagcaaga gattggcaaa 6540

gccaccgcca agtacttctt ctactctaat atcatgaatt tctttaagac tgagataacc 6600

ctggctaacg gcgaaatccg gaagcgccca ctgatcgaaa caaacggaga aacaggagaa 6660

atcgtgtggg ataaaggcag ggacttcgca actgtgcgga aggtgctgtc catgccacaa 6720

gtcaatatcg tgaagaagac cgaagtgcag accggcggat tctcaaagga gagcatcctg 6780

ccaaagcgga actctgacaa gctgatcgcc aggaagaaag attgggaccc aaagaagtat 6840

ggcggtttcg attcccctac agtggcttat tccgttctgg tcgtggcaaa agtggagaaa 6900

ggcaagtcca agaaactcaa gtctgttaag gagctgctcg gaattactat tatggagaga 6960

tccagcttcg agaagaatcc aatcgatttc ctggaagcta agggctataa agaagtgaag 7020

aaagatctca tcatcaaact gcccaagtac tctctctttg agctggagaa tggtaggaag 7080

cggatgctgg cctccgccgg agagctgcag aaaggaaacg agctggctct gccctccaaa 7140

tacgtgaact tcctgtatct ggcctcccac tacgagaaac tcaaaggtag ccctgaagac 7200

aatgagcaga agcaactctt tgttgagcaa cataaacact acctggacga aatcattgaa 7260

cagattagcg agttcagcaa gcgggttatt ctggccgatg caaacctcga taaagtgctg 7320

agcgcatata ataagcacag ggacaagcca attcgcgaac aagcagagaa tattatccac 7380

ctctttactc tgactaatct gggcgctcct gctgccttca agtatttcga tacaactatt 7440

gacaggaagc ggtacacctc taccaaagaa gttctcgatg ccaccctgat acaccagtca 7500

attaccggac tgtacgagac tcgcatcgac ctgtctcagc tcggcggcga cggttctccc 7560

aagaagaaga ggaaagtctc gaccggtgga gctgcaggaa tggtgagcaa gggcgaggag 7620

ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 7680

ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 7740

atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 7800

ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 7860

gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 7920

aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 7980

ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 8040

agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 8100

atccgccaca aca 8113

<210>77

<211>5294

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>77

ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60

cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120

ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180

cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240

tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300

tgtggaaagg acgaaacacc gtgagaccga gagagggtct cagttttaga gctagaaata 360

gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 420

tttttaaaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg 480

ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt 540

gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg 600

actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt 660

ggaaaggacg aaacaccggg tcttcgagaa gacctgtttt agagctagaa atagcaagtt 720

aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt 780

ttagaattct cgacctcgag acaaatggca gtattcatcc acaattttaa aagaaaaggg 840

gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa 900

actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac 960

agcagagatc cactttggcc gcggctcgag ggggttgggg ttgcgccttt tccaaggcag 1020

ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc 1080

cgaccctggg actcgcacat tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc 1140

tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt 1200

gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc 1260

agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg tggccaatag 1320

cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc gggaggcggg 1380

gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc 1440

tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc tctctcccca 1500

gggggatcca ccggagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg 1560

cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac 1620

gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt 1680

cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt 1740

ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc 1800

gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct 1860

ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga 1920

ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg 1980

cgccggggtg cccgccttcc tggaaacctc cgcgccccgc aacctcccct tctacgagcg 2040

gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat 2100

gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag 2160

cgcacgaccc catgcatcgg tacctttaag accaatgact tacaaggcag ctgtagatct 2220

tagccacttt ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg 2280

atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt 2340

gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca 2400

attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt 2460

aaaacctcta caaatgtggt aaaatcgata aggatccgtc gaccgatgcc cttgagagcc 2520

ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttttt 2580

atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc 2640

gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 2700

gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 2760

ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 2820

cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 2880

ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 2940

taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg 3000

ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 3060

ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 3120

aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 3180

tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac 3240

agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 3300

ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 3360

tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 3420

tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 3480

cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 3540

aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 3600

atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 3660

cttaccatct ggccccagtg ctgcaatgat accgcgggac ccacgctcac cggctccaga 3720

tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 3780

atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 3840

taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 3900

tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 3960

gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 4020

cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 4080

cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 4140

gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 4200

aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 4260

accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 4320

ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 4380

gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 4440

aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 4500

taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag 4560

cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 4620

cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 4680

tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 4740

cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 4800

gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 4860

aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc 4920

gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 4980

caaaatatta acgtttacaa tttcccattc gccattcagg ctgcgcaact gttgggaagg 5040

gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat aagtaagtaa 5100

tattaaggta cgggaggtac ttggagcggc cgcaataaaa tatctttatt ttcattacat 5160

ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca tcaaaacaaa 5220

acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg tgccagaaca 5280

tttctctatc gata 5294

Claims (10)

1. A sgRNA for targeted knockout of a human NKG2A/K L RC1 gene, wherein the nucleotide sequence of the sgRNA is shown in any one of SEQ ID NO. 1-56.

2. The sgRNA used for targeted knockout of the human NKG2A/K L RC1 gene according to claim 1, wherein the method for designing the nucleotide sequence of the sgRNA comprises the following steps:

s1, selecting common exons of different splicing forms on an NKG2A/K L RC1 gene;

s2, finding out the sequence of 5 '-GGN (19) GG-3', 5 '-GN (20) GG-3' or 5 '-N (21) GG-3' in the common exon after the first initiation codon ATG.

3. The sgRNA for targeted knockout of the human NKG2A/K L RC1 gene according to claim 2, wherein the sequence does not comprise more than 3 consecutive bases A or T.

4. The sgRNA for targeted knockout of the human NKG2A/K L RC1 gene according to claim 2, wherein the sequence is a single sequence or a pair of sequences, and the pair of sequences are separated by a distance of 0-200 bp.

5. An expression vector for expressing the sgRNA of any one of claims 1 to 4 for targeted knockout of the human NKG2A/K L RC1 gene.

6. A CRISPR/Cas9 system comprising sgRNA of any one of claims 1 to 4 for targeted knockout of human NKG2A/K L RC1 gene, further comprising a Cas9 protein expression plasmid.

7. A kit for targeted knockout of sgRNA of a human NKG2A/K L RC1 gene, which is characterized by comprising a vector for expressing the sgRNA and a Cas9 protein, wherein the nucleotide sequence of the sgRNA is shown as any one of SEQ ID NO. 1-56.

8. A sgRNA composition for targeted knockout of the human NKG2A/K L RC1 gene, characterized in that the sgRNA composition comprises at least two of the sgRNAs with the nucleotide sequences of SEQ ID No. 1-56.

9. The use of the sgRNA of any one of claims 1 to 4 for targeted knockout of the human NKG2A/K L RC1 gene in the preparation of an immune cell drug for treating a tumor disease.

10. Use of the sgRNA composition of claim 8 for targeted knockout of the human NKG2A/K L RC1 gene in the preparation of an immune cell medicament for treating a tumor disease.

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