CN109735540B

CN109735540B - SH2D1A gene, sgRNA and application thereof

Info

Publication number: CN109735540B
Application number: CN201910057956.8A
Authority: CN
Inventors: 孙倍成; 何其峰; 王晓琛; 黄新立; 夏安亮
Original assignee: Nanjing Drum Tower Hospital
Current assignee: Nanjing Drum Tower Hospital
Priority date: 2019-01-22
Filing date: 2019-01-22
Publication date: 2022-05-31
Anticipated expiration: 2039-01-22
Also published as: CN109735540A

Abstract

The invention discloses an application of an SH2D1A gene and sgRNA thereof in preparation of a medicine for recovering T cell exhaustion or an antitumor medicine, and relates to the field of T cell molecular biology; according to the invention, through discovery, detection and application of genes with the most significant expression difference in CD8+ T cells with different exhaustion degrees, SH2D1A is found to have an important regulation effect in the occurrence and development of CD8+ T cell exhaustion through high-throughput RNA sequencing, and SH2D1A expression is closely related to the occurrence and development of CD8+ T cell exhaustion; and then, sgRNA is designed and synthesized according to an SH2D1A coding sequence, and through combination and transformation with a plasmid vector, SH2D1A is knocked down in CD8+ T cells through a CRISPR/Cas9 gene editing technology, so that the occurrence of CD8+ T cell exhaustion can be obviously reduced, the anti-tumor capacity of the exhausted CD8+ T cells is obviously recovered, and the sgRNA can be applied to preparation of anti-tumor drugs.

Description

SH2D1A gene, sgRNA and application thereof

Technical Field

The invention belongs to the field of T cell molecular biology, and particularly relates to an SH2D1A gene, an sgRNA thereof and application thereof.

Background

Primary liver Cancer is one of the most common malignant tumors in China, the incidence rate of the primary liver Cancer is fifth in the world, the fatality rate is third in the line, and the primary liver Cancer can be divided into three types, namely a hepatocyte type, a cholangiocyte type and a mixed type according to pathological typing, wherein the primary liver Cancer (HCC) is the most common and accounts for more than 90 percent of the primary liver Cancer (Chen W, et al. Cancer statistics in China,2015.CA Cancer J Clin 2016; 66: 115-.

The main causes of HCC are chronic active hepatitis, including viral hepatitis caused by Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV), alcoholic hepatitis and non-alcoholic fatty liver hepatitis. At present, the common liver cell liver cancer in China takes HBV infection as a main approach. HCC is mainly characterized by early symptom hiding, high recurrence rate and poor prognosis (Llovet JM, et al. Advances in targeted therapeutics for hepatocellular receptors in the genetic era. Nat Rev Clin Oncol 2015; 12: 408. J. Hepatol 2015; Miltiadous O, et al. Progeneticor cell markers for polypeptides with hepatocellular receptors and Milan undersize expression 1377; Zhu AX, et al. HCC and genetics: porous target and future therapeutics Clin col 301; 8: 292. J. Oncol.). Hetu et al. HCC. and genetics. HCC. and prognostics. Nat. Clin Oncol 301). The current treatment methods of HCC mainly comprise surgical treatment, radiotherapy, interventional therapy, chemotherapy and the like. Immunotherapy, which has been activated in recent years, achieves the goal of resisting tumors by regulating the immune system of the body, improving the killing capability of the immune system to tumor cells and improving the immune suppression state of tumors, is a promising development direction of tumor therapy because of playing an important role in the treatment of various malignant tumors, and has related research and reports on its application in the treatment of HCC (GreenTF, et al. Current receptors of animal based tumors for patients with HCC: om basic science to novel tumor therapy approach. gut 2015; 64: 842. 848; Prieto J, et al. Immunoglial landscapes and immunotherapy of pathological cellular therapy. Nat. Rev stress liver 681; 12: Immunoglutal. 700-).

The immune system plays an extremely important and complex role in the transformation of chronic inflammation into tumors, and a special T cell dysfunction phenomenon, called T cell exhaustion (T cell ablation), is ubiquitous in the tumor microenvironment. The exhausted T cells mainly have weakened cell activity, proliferation capacity and anti-apoptosis capacity, and reduced capacity of secreting effector cytokines (IL-2, IFN-gamma, TNF and the like) and killing capacity. In a chronic inflammatory environment, a low-level immune state mediated by T cell exhaustion provides potential for tumorigenesis, and in the tumorigenesis and development process, the worsening of the T cell exhaustion degree gradually leads to the weakening of anti-tumor immunity, so that the tumor immune microenvironment becomes more suitable for tumor development. Depleted T cells express a series of immunosuppressive receptors on the surface, such as PD-1(programmed cell death-1), CTLA-4(cytoSH2D1Aic T-lymphocyte antigen-4), LAG-3(lymphocyte-activation gene-3), TIM-3(T cell immunologlobulin and mucin-domain-binding molecule-3), (Kim PS, et al.

T cell depletion, which is divided into reversible early depletion (moderate expression level of PD-1 on the surface of T cells) and irreversible late depletion (very high expression level of PD-1 on the surface of T cells) (where the expression level of PD-1 on the surface of T cells is high) (where the expression level of PD-J is high, et al. Molecular and cellular tissues is high in T cell expression. Nat Rev Immunol 2015; 15: 486-. Treatment with blocking T cell surface inhibitory molecules against early depleted T cells restored T cell function to some extent (where the term EJ, et al. molecular and cellular antigens in T cell expression. Nat Rev Immunol 2015; 15:486 499; Fisero P, et al. anti-inflammatory and cellular antigens T-cell expression can be restored by blocked expressed by marked expression-1 pathway in viral hepatitis B. gateway 2010; 138:682 693, 2006 e 681-684; Day CL, et al. PD-1expression HIV-specific T cell expression with T-cell expression, 53: 53. native expression, 53. DL; 78. expression, 354. native II. PD-1 expression. However, this approach can only partially improve the function of early depleted T cells and has little effect on reversing the late depleted T cell state. This may be the important reason why many tumor patients are not sensitive to treatment with antibodies such as PD-1, CTLA-4, etc. in clinical treatment, and therefore, the development of new highly effective antitumor drugs that specifically hinder the development of effector T cells to late exhaustion and restore the function of these late exhausted T cells has become a technical problem to be solved urgently in the art for the research of irreversible late T cell exhaustion.

The current mechanistic studies on T Cell exhaustion have found a plurality of molecules with important regulatory effects in the development of T Cell exhaustion, such as PSGL-1, NFAT, Blimp-1, TNF, GSK3, etc., suggesting that these molecules can be used as potential therapeutic targets for reversing T Cell exhaustion (Tinoco R, et al, PSGL-1Is an Immune chemistry Regulator T Cell Exception. 2016; 44: 1190-1203.14; Taylor A, et al. Glycogen Synthase Kinase 3 Infection drive T-beta-Mediated differentiation of Co-receptor PD-1to Enhance CD8 Cytolytic T Cell Impulse 2016; 44:274,278. 35. mu. F42. gamma.),278. mu. 18. mu. J.: Na + 42. mu. J.),278. T Cell Exception. mu. 18. mu. 42. J.),278, a role for the translating decompressor Blimp-1in CD8(+) T cell expansion reducing respiratory viral infection. Immunity 2009; 31: 309-320; huang X, et al, driving an improved CAR for cancer immunology.j Clin Invest 2016; 126: 2795-; qin H, et al, Generation of a new therapeutic peptide that is derived from a bacterial cell in a molecular-bearing die Nat Med 2014; 20: 676-; rosenberg SA, et al, adaptive cell transfer as personalised immunology for human cancer. science 2015; 348: 62-68; thorne SH, et al.synergistic inhibitors effects of immune cell-viral biotype, Science 2006; 1780 and 1784; van der Stegen SJ, Hamieh M, Sadelain M. the pharmacological of second-generation polymeric antigen receptors. nat Rev Drug Discov 2015; 14:499-509.). However, the specific molecular mechanism of T depletion is still far from being elucidated, and the way to develop anti-tumor therapies by interfering with T cell depletion is still difficult and lengthy.

The SH2D1A Gene (Gene located on human X chromosome q25, 2523bp in full length, encoding 129 amino acids, protein cytoplasmic expression, NCBI Gene ID:4068) was reported to be involved in regulating the activation signal of lymphocyte activating molecules, acting as an activating molecule inhibitor by blocking the recruitment of SH2 domain-containing signaling molecule SHP-2 to the binding site. Sh2d1a may also regulate signal transduction pathways in cells by binding to other surface molecules expressed on activated T, B and NK cells. Previous studies reported that Sh2d1a mutation caused uncontrolled activation of the immune system leading to systemic high inflammation; another study showed that decreased expression of Sh2D1a protein in T cells promotes continued activation of T cells leading to systemic lupus erythematosus (DE LA VARGA-MARTINEZ R, MORA-LOPEZ F, GARCIA-CUESTA D, et al.X-linked proliferative Disease Type 1in a Patient With the p.Gly93Asp SH2D1A Gene Mutation and hepatogenic cytological Lymphohistiocytosis [ J ]. Journal of diagnostic hematology/oncology,2017,39(8): e483-e 5). A study by Morra M et al showed that SH2D1A deficiency results in B cell dysfunction (Morra M, BARRINGTON R A, ABADIA-MOLINA A C, et al. defective B cell responses in the presence of SH2D1A [ J ]. Proceedings of the National Academy of Sciences of the United States of America,2005,102(13): 4819-23.). At present, the application of the SH2D1A gene in restoring CD8+ T cells infiltrated and exhausted in liver cancer tissues is not reported.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems, the applicant conducts expression profile gene chip analysis, finds the key role of the SH2D1A (SH2 domain relating 1A, SH2D1A) gene in promoting the T cell exhaustion in liver cancer, and further provides the application of the gene and the sgRNA thereof in preparing a medicine for restoring the T cell exhaustion or preparing a medicine for treating the liver cancer.

The invention firstly provides application of an SH2D1A gene or sgRNA thereof in preparation of a medicine for restoring T cell exhaustion or an anti-tumor medicine.

Further, the T cells are preferably CD8+ cells which are infiltratively depleted in the liver cancer tissue.

Further, the nucleotide sequence of the sgRNA is selected from:

1) any one of the nucleotide sequences shown as SEQ ID NO.3-SEQ ID NO. 17;

2) substituting and/or deleting and/or adding one or more bases of the nucleotide sequence in the 1) into the nucleotide sequence with the same functions as the nucleotide sequence in the 1).

Specifically, the application refers to: designing an exon 3(exon3) sequence of a coding sequence, and detecting 5 sgRNA sequences for guiding Cas9 enzyme to specifically shear an SH2D1A coding sequence and inhibiting SH2D1A expression, wherein the sequences are as follows: SH2D1A-sgRNA1 (the nucleotide sequence of which is shown in SEQ ID NO. 3), SH2D1A-sgRNA2 (the nucleotide sequence of which is shown in SEQ ID NO. 4), SH2D1A-sgRNA3 (the nucleotide sequence of which is shown in SEQ ID NO. 5), SH2D1A-sgRNA4 (the nucleotide sequence of which is shown in SEQ ID NO. 6) and SH2D1A-sgRNA5 (the nucleotide sequence of which is shown in SEQ ID NO. 7).

Specifically, the application further comprises: synthesizing SH2D1A-sgRNA Oligo 1-SH 2D1A-sgRNA Oligo5 sequences with adhesive ends by adding SH2D1A-sgRNA 1-SH 2D1A-sgRNA5 sequences; the sequence is shown in SEQ ID NO.8-SEQ ID NO. 17;

the invention also includes DNA molecules encoding the sgrnas.

The invention also includes expression cassettes, recombinant vectors or cells containing the sgrnas or the DNA molecules.

The invention also discloses application of the sgRNA in SH2D1A gene knockout.

The invention also discloses a method for targeted specific knockout of SH2D1A gene in infiltrative CD8+ T cells in liver cancer tissues by using the CRISPR/Cas9 gene editing technology, which comprises the following steps:

1) design of SH2D 1A-sgRNA: according to the gene sequence of SH2D1A and PAM sequence positioning, 5 sites on SH2D1A exon3 are selected to design the nucleotide sequence of sgRNA; the sequence is shown in any one of SEQ ID NO.3-SEQ ID NO. 7;

2) synthesis of SH2D1A-sgRNA Oligo: synthesizing an SH2D1A-sgRNA Oligo sequence added with a cohesive end according to the designed sgRNA sequence; the sequence is shown in any one of SEQ ID NO.8-SEQ ID NO. 17;

3) connection of SH2D 1A-sgRNA: carrying out restriction enzyme digestion on pGL3-U6-sgRNA-PGK-puromycin vector plasmid by using a restriction enzyme BsaI, and then connecting and synthesizing SH2D1A-sgRNA Oligo sequence double chains with cohesive ends through solutionI;

4) transformation of the plasmid: transforming the ligation product obtained in the step 3) into escherichia coli to obtain a monoclonal antibody, and extracting to obtain a plasmid;

6) in vitro transcription of plasmids: carrying out reverse transcription on the plasmid to obtain the target SH2D 1A-sgRNA;

6) and electrically transfecting the Cas9 protein and the SH2D1A-sgRNA to obtain the SH2D 1A-specifically knocked-out CD8+ depleted T cells.

The invention also comprises a construction method of the SH2D1A gene-deleted cell strain, which comprises the step of carrying out subculturing and screening on the SH2D 1A-knocked out CD8+ depleted T cells to obtain stable SH2D 1A-knocked out CD8+ depleted T cells.

The invention also comprises an SH2D1A gene-deleted cell strain obtained by the construction method.

The invention also comprises a kit for SH2D1A gene knockout, wherein the kit comprises any one of the following components i to iii:

i. the nucleotide sequence of the sgRNA of claim;

ii. The DNA molecule, the expression cassette, the recombinant vector or the cell as described;

iii, SH2D1A gene-deleted cell line as described.

Specifically, the invention also provides a method for designing sgRNA by using the SH2D1A gene and specifically knocking out the SH2D1A gene in invasive CD8+ T cells in liver cancer tissues in a targeted manner by using a CRISPR/Cas9 gene editing technology, which comprises the following steps:

1) design of SH2D 1A-sgRNA: according to the Gene sequence of SH2D1A on NCBI Gene (NCBI Gene ID:4068), in combination with the CRISPR/Cas9 Gene editing principle, according to PAM sequence positioning, selecting 5 sites on SH2D1A exon3, and designing a multi-segment sgRNA sequence, wherein the designed nucleotide sequence is shown as SEQ ID NO.3-SEQ ID NO. 7.

2) Synthesis of SH2D 1A-sgRNA: combining the characteristics of pGL3-U6-sgRNA-PGK-puromycin carrier plasmid (containing a U6 promoter and capable of quickly expressing sgRNA in eukaryotes) and restriction enzyme BsaI (NEB, R3535S), adding sticky end Oligo sequences to the sgRNAs designed in the step 1) respectively, and synthesizing SH2D1A-sgRNA, wherein the sequences are shown in SEQ ID NO.8-SEQ ID NO. 17.

3) SH2D1A-sgRNA ligation: annealing each pair of SH2D1A-sgRNA synthesized in the step 2), linearizing pGL3-U6-sgRNA-PGK-puromycin vector plasmid, cutting with restriction enzyme BsaI (NEB, R3535S), and connecting synthesized SH2D1A-sgRNA double strand with adhesive end through solvation I (TAKARA).

4) Transformation of the plasmid: using E.coli, by transformation

pGL3-U6-SH2D1AsgRNA-PGK-puromycin plasmid, screening positive large intestinal bacillus monoclonal by ampicillin, further amplifying target product and extracting plasmid.

5) In vitro transcription of the plasmid: by MEGAshortscript^TMKit (thermo Fisher scientific) performed in vitro transcription amplification of sgRNA on the extracted plasmids.

6) Cas9 protein and SH2D1A-sgRNA electrotransfection: cells were electroporated using a Lonza 4D-Nucleofector electrotransfer instrument:

the electric rotating body is: cas9 protein 400ng + sgRNA 200ng +2 x 10^5 cells + P3 Primary Cell 4D-Nucleofector^TM X Kit(Lonza)；

Electric rotating mode: human T cell.

The inventors discovered a new gene determining the depletion of CD8+ T cells by systematically studying the transcriptome expression profile of infiltrating CD8+ T cells in human liver cancer tissues, and sorted the infiltrating CD8+ T cells in liver cancer tissues according to the expression level of cell surface inhibitory surface receptors (PD-1, TIM3) by flow cytometry: 3 cases of non-exhausted CD8+ T cells (PD1-TIM3-), 3 cases of exhausted CD8+ T cells (PD1+ TIM3+), and 6 samples in total, then through high-throughput RNA sequencing, a group of genes which are differentially expressed in CD8+ T cells with different exhaustion degrees are found, and further through a real-time quantitative PCR technology, the genes which are most significantly differentially expressed in the exhausted CD8+ T cells are verified in the non-exhausted and exhausted CD8+ T cells of liver cancer tissues of 40 patients, and SH2D1A which is one of the genes with the most significant expression difference in the exhausted CD8+ T cells is selected. In the examples, the applicant further determined that SH2D1A has an important regulatory role in the development of CD8+ T cell depletion by overexpressing SH2D1A in non-depleted CD8+ T cells and knocking down SH2D1A in depleted CD8+ T cells, and that reducing SH2D1A expression can significantly reduce the development of CD8+ T cell depletion and significantly restore the anti-tumor capacity of depleted CD8+ T cells. Therefore, the SH2D1A gene is expected to become a new target point of liver cancer immunotherapy, thereby providing help for inhibiting the occurrence and development of liver cancer and improving the prognosis of liver cancer patients.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1) the invention discovers for the first time that the SH2D1A gene has an obvious effect on recovering the inhibitory effect of CD8+ T cell tumor which is infiltrated and exhausted in liver cancer tissues.

2) The invention designs the sgRNA sequence of SH2D1A for the first time, successfully knocks out SH2D1A gene in invasive CD8+ T cells in liver cancer tissues, and obtains exhausted CD8+ T cells with specific knockout of SH2D 1A.

3) The invention successfully proves that the SH2D1A knocked-out CRISPR/Cas9 with adoptive therapy in mice has obvious anti-tumor effect on reversely exhausted CD8+ T cells for the first time.

Drawings

FIG. 1is a schematic diagram of pGL3-U6-sgRNA-PGK-puromycin vector;

figure 2 is a schematic diagram of flow identification of depleted and non-depleted CD8+ T cells; isotype is an Isotype control and used in flow assays as a blank control. (PD1-TIM3-) non-exhausted CD8+ T cells, (PD1+ TIM3+) exhausted CD8+ T cells, and compared with the non-exhausted CD8+ T cells, the activity (CD69) of the exhausted CD8+ T cells, the proliferation level (Ki67) and the level of cytokines (IL-2, IFN-gamma and TNF-alpha) are all obviously reduced;

figure 3 is a schematic diagram of flow sorting of depleted and non-depleted CD8+ T cells;

FIG. 4 is a schematic representation of qPCR detection of mRNA expression of the SH2D1A gene in depleted and non-depleted CD8+ T cells;

FIG. 5 is an agarose gel electrophoresis of the cleavage effect of 5 SH2D 1A-specific sgRNAs; in the figure, lanes 1-9 are marker, sgRNA1, sgRNA2, sgRNA3, control, sgRNA4, sgRNA5, control and marker in turn;

FIG. 6 is a schematic diagram of expression of SH2D1AmRNA in depleted CD8+ T cells 1-5 after detection of CRISPR/Cas9 knockout by qPCR;

FIG. 7 is a schematic diagram showing the expression condition of SH2D1A protein in exhausted CD8+ T cells 1-5 after detecting CRISPR/Cas9 knockout by western blot;

fig. 8 is a schematic diagram of flow cytometry detection of SH2D1A protein expression in CD8+ T cell 3 depleted after CRISPR/Cas9 knockout;

fig. 9 is a schematic diagram of flow cytometry detection of CD8+ T cell 3 proliferation activity depleted following CRISPR/Cas9 knockout;

fig. 10 is a schematic diagram of flow cytometry detection of CD8+ T cell 3 cytokine depleted following CRISPR/Cas9 knockout and depletion-associated molecules;

FIG. 11 is a photograph of in vitro NCG mouse subcutaneous hybridoma assay for detecting CRISPR/Cas9 knockout SH2D1A depleted CD8+ T cell 3 anti-tumor capacity;

fig. 12 is a schematic diagram of sgRNA double strands with enzyme-linked sites added at both ends (from top to bottom, corresponding to sgRNA1, sgRNA2, sgRNA3, sgRNA4, and sgRNA5, respectively);

fig. 13 is an agarose gel electrophoresis image of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, negative control, positive control, and DNA marker 2000.

Detailed Description

And (3) reagent sources: pGL3-U6-sgRNA-PGK-puromycin purchased from addgene

PrimeScript RT Master Mix, solutionI were purchased from Takara;

ampicillin screening positive escherichia coli is purchased from organisms of the department of Onychidae;

TransScript Two-Step RT-PCR SuperMix was purchased from TRANSGEN;

AxyPrep Plasmid Miniprep Kit was purchased from Axygen;

MEGAshortscript^TMkit was purchased from ThermoFisher corporation;

the source of the sample: all CD8+ T cells and the like are provided by Nanjing drumbeat hospital;

EXAMPLE 1 screening of SH2D1A Gene

The applicant systematically studied the transcriptome expression profile of infiltrating CD8+ T cells in human liver cancer tissues after the project was reviewed by the ethics committee, found a new gene that determines CD8+ T cell depletion, and screened liver cancer tissue infiltrating CD8+ T cells according to the expression level of cell surface inhibitory surface receptors (PD-1, TIM3) by flow cytometry: 3 non-exhausted CD8+ T cells (PD1-TIM3-), 3 exhausted CD8+ T cells (PD1+ TIM3+), and 6 samples (derived from 3 patients with specific information shown in Table 1):

table 1 sample information 1

Table 2 sample information 2

Through high-throughput RNA sequencing, a group of genes differentially expressed in CD8+ T cells with different exhaustion degrees are discovered, and further, through the verification of a real-time quantitative PCR technology in non-exhausted, partially-exhausted and completely-exhausted CD8+ T cells of liver cancer tissue infiltration of 40 patients (see the specific information in Table 2), one of the genes with the most significant expression difference in CD8+ T cells with different exhaustion degrees is selected as SH2D 1A.

The method comprises the following specific steps:

1) sorting of differently depleted CD8+ T cells: invasive CD8+ T cells from liver cancer tissues were sorted 40 by flow cytometry (BD Biosciences) into two categories based on surface marker expression: non-exhausted CD8+ T Cells (PD1-TIM3-), exhausted CD8+ T Cells (PD1+ TIM3+) (Zhen et al, Targeting type I interaction-mediated Activation reactions functions in chronic HIV infection. J Clin invest.2017; 127(1): 260-268; Singer et al, A Distingt Gene Module for Dysfunction Unbound efficiency in far-isolating T Cells 2016, Cell 166, 1500-Cell 1511).

The results of the experiment are shown in fig. 2 and 3: figure 2 is a schematic representation of flow identification of depleted and non-depleted CD8+ T cells. Isotype is an Isotype control and used in flow assays as a blank control. (PD1-TIM3-) non-exhausted CD8+ T cells, (PD1+ TIM3+) exhausted CD8+ T cells, compared with the non-exhausted CD8+ T cells, the activity (CD69) of the exhausted CD8+ T cells, the proliferation level (Ki67) and the level of cell factors (IL-2, IFN-gamma and TNF-alpha) are all obviously reduced.

Figure 3 is flow sort depleted and non-depleted CD8+ T cells.

2) Total RNA extraction

Isolated CD8+ T cells were lysed by trizol (invitrogen), followed by extraction of total RNA using chloroform, isopropanol, and 75% ethanol.

See methods for total RNA extraction: (Brown, R.A.M., et al. (2018). "Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kit area accessed. RTM. Biotechnol 18(1):16.)

3) Preparation of cDNA

cDNA was prepared by reverse transcription reagent PrimeScript RT Master Mix.

Reaction conditions are as follows: 15min at 37 ℃, 5sec at 85 ℃ and infinity at 4 ℃

The reaction system is as follows:

4) qPCR detection of SH2D1A mRNA expression in depleted and non-depleted CD8+ T cells

Designing a specific primer (the nucleotide sequence of which is shown in SEQ ID NO.18 and SEQ ID NO.19) according to the SH2D1A mRNA sequence condition;

fluorescent quantitative PCR procedure: pre-denaturation at 95 ℃ for 30s, followed by 40 cycles: 95 ℃ for 5s, 60 ℃ for 32 s.

The reaction system is as follows:

the results of the experiment are shown in FIG. 4: fig. 4 is a graph showing the mRNA expression of the SH2D1A gene in depleted and non-depleted CD8+ T cells by qPCR, showing that SH2D1AmRNA is overexpressed in depleted CD8+ T cells compared to non-depleted CD8+ T cells. In the figure, PD1-TIM 3-is a non-depleted CD8+ T cell, PD1^-TIM3+ is a depleted CD8+ T cell.

Example 2 design, Synthesis, ligation, transformation, electrotransfection of sgRNAs

1) Design of SH2D1A-sgRNA

According to the gene sequence (the sequence is shown as SEQ ID NO. 1) of SH2D1A on NCBI Genbank, the CRISPR/Cas9 gene editing principle is combined, according to PAM sequence (NGG) positioning and according to exon3 (the sequence is shown as SEQ ID NO. 2), 5 sites on SH2D1A exon3 are selected, sgRNA sequences 1-5 are designed, and the nucleotide sequences are sequentially shown as SEQ ID NO.3-SEQ ID NO. 7.

2) Synthesis of SH2D1A-sgRNA

Binds pGL3-U6-sgRNA-PGK-puromycin vector plasmid (the vector structure is shown in FIG. 1) (Carleton, J.B., et al (2017). "Multiplex Enhancer Interference vectors colloidal Control of Gene Regulation by Estrogen Receptitor alpha-Bound Enhancers"Cell Syst333-344e335.) and the characteristics of restriction enzyme BsaI (NEB, R3535S), on the basis of sgRNA 1-5 designed in step 1), SH2D1A-sgRNA Oligo sequences (named Oligo 1-Oligo 5 respectively, the nucleotide sequences of which are shown in SEQ ID NO.8-SEQ ID NO.17 in sequence) are synthesized, wherein SEQ ID NO.8(Up Oligo1) and SEQ ID NO.9 (Down Oligo1), SEQ ID NO.10(Up Oligo2) and SEQ ID NO.11(Down Oligo2), SEQ ID NO.12(Up Oligo3) and SEQ ID NO.13(Down Oligo3), SEQ ID NO.14 (Up Oligo4) and SEQ ID NO.15(Down Oligo 53982), and SEQ ID NO. 4) with cohesive ends addedBoth NO.16(Up Oligo4) and SEQ ID NO.17(Down Oligo5) are reverse complementary strands.

3) Ligation of SH2D1A-sgRNA

pGL3-U6-sgRNA-PGK-puromycin vector plasmid is cut with restriction enzyme BsaI (NEB, R3535S), and then is connected with synthesized SH2D1A-sgRNA double chain with a cohesive end through solutionI (the nucleotide sequence is shown in FIG. 12).

The method comprises the following specific steps:

a) annealing each pair of the synthesized oligos to obtain an annealed product.

The annealing system is:

4.5μl Up Oligo(100μM)

4.5μl Down Oligo(100μM)

1μl NEB buffer 2

run in a PCR instrument (applied biosystems Thermo Fisher Scientific) according to the following touch down program:

95℃，5min；

95–85℃at-2℃/s；

85–25℃at-0.1℃/s；

hold at 4℃.

b) linearized pGL3-U6-sgRNA-PGK-puromycin plasmid

3μg pGL3-U6-sgRNA-PGK-puromycin

5μl CutSmart Buffer

1.5μl BsaI(NEB，R3535S)

Adding water to 50 mu l, incubating for 3-4 hours at 37 ℃, shaking at intervals and centrifuging to prevent liquid drops from evaporating to a tube cover. The AxyPrep PCR clean Kit is used for purification and recovery into 20-40 mul of sterilized water, and the purification steps are strictly operated according to the Kit instructions.

c) Connecting the sgRNA vector to obtain pGL3-U6-SH2D1AsgRNA 1-5-PGK-puromycin plasmid

2 μ l of annealed product

Mu.l pGL3-U6-sgRNA plasmid linearization product (25 ng/. mu.l)

3μl solutionI(TAKARA)

Incubate at 16 ℃ for more than 1 hour.

4) Transformation of the plasmid: pGL3-U6-SH2D1AsgRNA 1-5-PGK-puromycin plasmid in vitro transcribes SH2D1A-sgRNA 1-5, and positive escherichia coli monoclonals are screened through ampicillin, and then target products are further amplified.

The method comprises the following specific steps:

a) pipetting 5-10. mu.l of the ligation product (from step 3) into 100. mu.l of competent cells (E.coli DH 5. alpha.), stirring well, and ice-cooling for 30 min;

b) water bath at 42 deg.C for 90 seconds, taking care not to shake the tube, ice for 2 min;

c) adding 800 μ L LB liquid (Tryptone 10g/L, Yeast extract 5g/L (Yeast extract) (Oxioid Ltd), sodium chloride 10g/L), shaking at 37 deg.C and 200rpm for 40-60 min;

d) centrifuging at 4500rpm for 5-10min, removing supernatant, collecting precipitate, homogenizing, and spot-coating on LB plate (Amp:100 μ g/ml, X-gal 40 μ l, IPTG 4 μ l) of X-gal + IPTG + Amp; standing at room temperature for 30min, and absorbing completely. Inverting the culture dish, and incubating at 37 deg.C for 12-16 hr;

e) picking a monoclonal colony, uniformly mixing the colony with 20 ul of LB culture medium, and carrying out colony PCR (polymerase chain reaction) by using an assembly Forward primer (SEQ ID NO.20) and synthetic Down oligos 1-5 (SEQ ID NO.9, 11, 13, 15 and 17) to identify TransScript Two-Step RT-PCR SuperMix;

RT-PCR procedure: the temperature of the mixture is 94 ℃ for 2min,

the specific reaction system is as follows:

f) mu.l of the above-mentioned bacterial clone was added to 15ml of LB medium containing Amp (100. mu.g/ml) at 37 ℃ in a shaker, shaken overnight and the Plasmid was extracted using AxyPrep Plasmid Miniprep Kit.

5) In vitro transcription of plasmids: using reagent MEGAshortscript^TMKit, obtaining the target SH2D1A-sgRNA 1-5. The method comprises the following specific steps:

a) adding RNA secure (ThermoFisher) into the extracted plasmid at 60 deg.C for 10min, and shaking for 1 time every 5 min;

b) recovering the clean;

c) 10 μ l System of transcription

Reaction conditions are as follows: 4h at 37 ℃ and 55 ℃ at cover;

reaction system:

RT-PCR procedure:

d) and (3) removing DNA: TURBO1 μ l, 37 ℃ 20 min.

e) FIG. 13 shows an agarose gel electrophoresis image.

f) RNA recovery and concentration measurement

The Elutionbuffer 100. mu.l + 350. mu.l Bindingbuffer + 250. mu.l ETOH mix was pre-mixed into the adsorption column and centrifuged at 1200rpm for 1min, 400. mu.l prep buffer 1200rpm for 30s, and 700. mu.l washingbuffer (containing ethanol) at 1200 rpm.

6) Cas9 protein and SH2D1A-sgRNA 1-5 electrotransfection: cells were electroporated using a Lonza 4D-Nucleofector electrotransfer instrument:

the electric rotating body is: cas9 protein (VAZYME EN301-01)400ng + sgRNA 1-5200 ng +2 x 10^5 cells + P3 Primary Cell 4D-Nucleofector^TM X Kit(Lonza)；

Electric rotating mode: human T cell.

The method comprises the following specific steps

a) CD8+ T cell counts, 2 x 10^5 cells per sample; centrifuging at 1000rpm for 5min, removing culture medium, adding PBS, resuspending, centrifuging at 1000rpm for 5min, removing PBS, adding electrotransfer buffer (P3 Primary Cell) 4D-Nucleofector^TMX Kit, Lonza) resuspension;

b) adding Cas9 protein 400ng + sgRNA 1-5200 ng, and adding an electric rotating cup after fully and uniformly mixing;

c) transfecting CD8+ T cells by using a Lonza 4D-Nucleofector electrotransfer instrument in a human T cell mode;

d) adding 100 μ l of a culture medium containing 10% FBS into the electric rotating cup, sufficiently blowing, and transferring the cells to a culture dish for continuous culture; thus obtaining the SH2D 1A-specific knockout CD8+ exhausted T cells 1-5.

Example 3 identification of SH2D1A Effect in Targeted specific knockout CD8+ T cells

1) RT-PCR method for detecting SH2D1AmRNA expression level in CD8+ T cells

a) Total RNA extraction

SH2D1A knock-out depleted CD8+ T cells 1-5 of example 2 were lysed by TRIzol (Invitrogen), followed by extraction of total RNA 1-5 using chloroform, isopropanol, and 75% ethanol.

b) Preparation of cDNA 1-5: preparation by reverse transcription reagent PrimeScript RT Master Mix (Takara)

cDNA reaction conditions: 15min at 37 ℃, 5sec at 85 ℃ and infinity at 4 ℃

The reaction system is as follows:

c) qPCR detection of SH2D1A mRNA expression in depleted and non-depleted CD8+ T cells

Specific primers were designed based on the SH2D1A mRNA sequence (SEQ ID NO.18 and SEQ ID NO.19)

The reaction system is as follows:

the results are shown in fig. 5 and 6:

FIG. 5 shows agarose gel electrophoresis of 5 SH2D 1A-specific sgRNA cleavage effects, wherein lanes 1-9 in the agarose gel are marker, sgRNA1, sgRNA2, sgRNA3, control, sgRNA4, sgRNA5, control, and marker, respectively.

Fig. 6 is a schematic diagram of qPCR detection of expression of 5 SH2D1A specific sgrnas in depleted CD8+ T cells following CRISPR/Cas9 knockout.

The above experiment results show that SH2D1A mrnas corresponding to sgRNA2, sgRNA3 and sgRNA4 of 5 SH2D 1A-sgrnas are all down-regulated, and the SH2D1A mRNA of sgRNA3 is most obviously down-regulated.

Next, we performed Westernblot experiments and flow cytometry, the specific experimental procedures were as follows:

westernblot experiment: the SH2D1A knockout exhaustive CD8+ T cells 1-5 in example 2 are respectively added with protein lysate RIPA (containing protease inhibitor and PMSF) to be cracked on ice for 30 minutes, centrifuged at 12000rpm for 10 minutes, the supernatant is taken, protein concentration is adjusted by a BCA method, and then 5x loading buffer is added, 95 ℃ and 10 minutes. And (3) loading 10 mu l of protein solution on polyacrylamide gel, incubating the primary antibody overnight after the blocking of the transferred membrane, incubating the secondary antibody for 2 hours after TBST is eluted for 3 times, and exposing after elution.

Flow cytometry: the SH2D1A (SH2D1A-sgRNA3) -knocked depleted CD8+ T cells 3 and control cells thereof are subjected to flow antibody staining, surface labeling of CD8, fixation of a membrane rupture intracellular labeling of SH2D1A, elution of unbound antibody by PBS, flow cytometry collection of the unbound antibody, and flowjo analysis of data.

Westernblot experiments and flow cytometry for detecting SH2D1A expression in CD8+ T cells, CD8+ T cell proliferation activity, cytokine and exhaustion related molecule expression after SH2D1A knockout are shown in FIGS. 7-10.

Fig. 7 is a schematic diagram of the western blot detection of the expression of SH2D1A protein in depleted CD8+ T cells after CRISPR/Cas9 knockout, and the results show that the SH2D1A protein expressions corresponding to sgRNA2, sgRNA3, and sgRNA4 groups in 5 SH2D 1A-sgrnas designed in the above example are all down-regulated, wherein the SH2D1A protein expression of the sgRNA3 group is most obviously down-regulated. Therefore, the sgRNA3 group SH2D1A protein is adopted in the subsequent flow cytometry detection experiments

Fig. 8 is a schematic diagram of flow cytometry for detecting the expression of SH2D1A protein in CD8+ T cells depleted after CRISPR/Cas9 knockout, wherein in the diagram, 1is NC (negative control group) 2 is SH2D1A-sgRNA3, and it can be seen that SH2D1A expression is significantly down-regulated after CRISPR/Cas9 knockout.

Fig. 9 is a graph showing that flow cytometry detects CD8+ T cell proliferation activity exhausted after CRISPR/Cas9 knockout, wherein the upper left graph (activity index CD69) and the upper right graph (activity index Ki67) show that CD8+ T cell proliferation activity indexes CD69 and Ki67 exhausted after SH2D1A knockout are significantly upregulated, and the lower left graph and the lower right graph are upper left and upper right histograms show that the difference between CD8+ T cell proliferation activity indexes CD69 and Ki67 exhausted after SH2D1A knockout is statistically significant upregulated. It can be seen that the proliferation level and activity of the exhausted CD8+ T cells are significantly up-regulated after SH2D1A is specifically knocked out.

Fig. 10 is a schematic diagram of flow cytometry for detecting CD8+ T cell cytokines and depletion-related molecules depleted after CRISPR/Cas9 knockout, wherein the upper left graph (cytokine IL2) and the upper right graph (cytokine IFN- γ) show that CD8+ T cell cytokines IL2, IFN- γ expression depleted after SH2D1A knockout is significantly up-regulated, and the lower left graph (cell depletion marker molecule TIM-3) and the lower right graph (cell depletion marker molecule PD-1) show that CD8+ T cell depletion marker molecules TIM-3 and PD-1expression depleted after SH2D1A knockout is significantly down-regulated. As can be seen, after SH2D1A is knocked out specifically, the expressions of exhausted CD8+ T cell cytokines IL2 and IFN-gamma are remarkably up-regulated, and the expressions of TIM-3 and PD-1 are remarkably down-regulated.

Example 4 mouse experiments

The experimental procedure was as follows:

1. selecting 8-10 weeks old NCG mice, inoculating the liver cancer cell line MHCC97H (purchased from cell bank of Chinese academy of sciences) on the back subcutaneously, and injecting 100 ten thousand cells per mouse subcutaneously;

after 2.1 weeks, CD8+ T cells (CD8+ T cells obtained in example 3 after SH2D1A is knocked out by sgRNA3) after SH2D1A is knocked out by CRISPR/Cas9 were injected into tail vein, and the size of back tumor of NCG mice was observed after two weeks/four weeks, respectively, with normal T cells as control group, and the anti-tumor ability of CD8+ T cells after SH2D1A is knocked out by CRISPR/Cas9 was evaluated.

The experimental results are shown in FIG. 11, in which group A (CD8+ + T cell-NC) is the control group, group B (CD8+ + T cell-SH2D1A-sgRNA) is the experimental group, group A has a tumor mass of 4.333 + -0.3584 g and a volume of 1464 + -120.5 mm³Compared with the group B, the mass is 1.843 +/-0.2852 g, and the volume is 494.2 +/-44.07 mm³The tumors in the group A are obviously increased, and the volume and the mass of the cells in the group B are obviously reduced by a lot compared with those in the group A, which shows that the anti-tumor capacity of the CD8+ T cells is obviously enhanced after SH2D1A is specifically knocked out.

It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only illustrative of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Nanjing drum building hospital

<120> SH2D1A gene, sgRNA and application thereof

<160>20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 378

<212> DNA

<213> Gene sequence of SH2D1A (SH2D1A)

<400> 1

atggacgcag tggctgtgta tcatggcaaa atcagcaggg aaaccggcga gaagctcctg 60

cttgccactg ggctggatgg cagctatttg ctgagggaca gcgagagcgt gccaggcgtg 120

tactgcctat gtgtgctgta tcacggttac atttatacat accgagtgtc ccagacagaa 180

acaggttctt ggagtgctga gacagcacct ggggtacata aaagatattt ccggaaaata 240

aaaaatctca tttcagcatt tcagaagcca gatcaaggca ttgtaatacc tctgcagtat 300

ccagttgaga agaagtcctc agctagaagt acacaaggga taagagaaga tcctgatgtc 360

tgcctgaaag ccccatga 378

<210> 2

<211> 136

<212> DNA

<213> exon3 of the gene sequence of SH2D1A (SH2D1A exon3)

<400> 2

acagcacctg gggtacataa aagatatttc cggaaaataa aaaatctcat ttcagcattt 60

cagaagccag atcaaggcat tgtaatacct ctgcagtatc cagttgagaa gaagtcctca 120

gctagaagta cacaag 136

<210> 3

<211> 20

<212> DNA

<213> sgRNA sequence 1(sgRNA1)

<400> 3

gcatccccgt ctgagtgcaa 20

<210> 4

<211> 20

<212> DNA

<213> sgRNA sequence 2(sgRNA2)

<400> 4

agcatccccg tctgagtgca 20

<210> 5

<211> 20

<212> DNA

<213> sgRNA sequence 3(sgRNA3)

<400> 5

tagacaacat cctgttgttg 20

<210> 6

<211> 20

<212> DNA

<213> sgRNA sequence 4(sgRNA4)

<400> 6

ctctgtatga accctgtgtt 20

<210> 7

<211> 20

<212> DNA

<213> sgRNA sequence 5(sgRNA5)

<400> 7

gtagacaaca tcctgttgtt 20

<210> 8

<211> 24

<212> DNA

<213> SH2D1A-sgRNA up Oligo1(SH2D1A-sgRNA up Oligo1)

<400> 8

accggcatcc ccgtctgagt gcaa 24

<210> 9

<211> 24

<212> DNA

<213> SH2D1A-sgRNA down Oligo1(SH2D1A-sgRNA down Oligo1)

<400> 9

aaacttgcac tcagacgggg atgc 24

<210> 10

<211> 24

<212> DNA

<213> SH2D1A-sgRNA up Oligo2(SH2D1A-sgRNA up Oligo2)

<400> 10

accgagcatc cccgtctgag tgca 24

<210> 11

<211> 24

<212> DNA

<213> SH2D1A-sgRNA down Oligo2(SH2D1A-sgRNA down Oligo2)

<400> 11

aaactgcact gagacgggga tgct 24

<210> 12

<211> 24

<212> DNA

<213> SH2D1A-sgRNA up Oligo3(SH2D1A-sgRNA up Oligo3)

<400> 12

accgtagaca acatcctgtt gttg 24

<210> 13

<211> 24

<212> DNA

<213> SH2D1A-sgRNA down Oligo3(SH2D1A-sgRNA down Oligo3)

<400> 13

aaaccaacaa caggatgttg tcta 24

<210> 14

<211> 24

<212> DNA

<213> SH2D1A-sgRNA up Oligo4(SH2D1A-sgRNA up Oligo4)

<400> 14

accgctctgt atgaaccctg tgtt 24

<210> 16

<211> 24

<212> DNA

<213> SH2D1A-sgRNA down Oligo4(SH2D1A-sgRNA down Oligo4)

<400> 16

aaacaacaca gggttcatac agag 24

<210> 16

<211> 24

<212> DNA

<213> SH2D1A-sgRNA up Oligo5(SH2D1A-sgRNA up Oligo5)

<400> 16

accggtagac aacatcctgt tgtt 24

<210> 17

<211> 24

<212> DNA

<213> SH2D1A-sgRNA down Oligo5(SH2D1A-sgRNA down Oligo5)

<400> 17

aaacaacaac aggatgttgt ctac 24

<210> 18

<211> 20

<212> DNA

<213> SH2D1A mRNA-specific Forward Primer (SH2D1A mRNA Forward Primer)

<400> 18

aggcgtgtac tgcctatgtg 20

<210> 19

<211> 23

<212> DNA

<213> SH2D1A mRNA-specific downstream Primer (SH2D1A mRNA Reverse Primer)

<400> 19

tgcagaggta ttacaatgcc ttg 23

<210> 20

<211> 23

<212> DNA

<213> assembly For primer (assembly For)

<400> 20

cgattagtga acggatctcg acg 23

Claims

The application of sgRNA of SH2D1A gene in preparing a medicine for restoring T cell exhaustion or preparing an anti-tumor medicine; the nucleotide sequence of the sgRNA is selected from:

1) any one of the nucleotide sequences shown in SEQ ID NO. 4-SEQ ID NO. 6.
2. An sgRNA, wherein the nucleotide sequence of the sgRNA is selected from the group consisting of:

1) any one of the nucleotide sequences shown in SEQ ID NO. 4-SEQ ID NO. 6.
3. A DNA molecule encoding the sgRNA of claim 2.
4. An expression cassette, recombinant vector or cell containing the sgRNA of claim 2 or the DNA molecule of claim 3.
5. A kit for SH2D1A gene knockout, which is characterized by comprising any one of the following i-ii:

i. the nucleotide sequence of the sgRNA of claim 2;

ii. The DNA molecule of claim 3, the expression cassette of claim 4, a recombinant vector or a cell.