CN108103025B

CN108103025B - Hematopoietic stem cell and preparation method and application thereof

Info

Publication number: CN108103025B
Application number: CN201711258136.2A
Authority: CN
Inventors: 祝海宝; 刘方方; 唐忆琳; 罗思施; 黄雨亭; 陶米林; 梁福才
Original assignee: Guangdong Chimeng Medical Technology Co ltd
Current assignee: Guangdong Chimeng Medical Technology Co ltd
Priority date: 2016-12-28
Filing date: 2017-12-01
Publication date: 2021-04-27
Anticipated expiration: 2037-12-01
Also published as: CN108103025A

Abstract

The invention provides a hematopoietic stem cell, wherein the expression level of ZBTB7A gene of the hematopoietic stem cell is reduced or not expressed. According to the invention, the CRISPR technology is adopted to knock out the fetal hemoglobin gene inhibitor, so that the operation is simple and convenient, and the time consumption is short; the fetal hemoglobin gene inhibitor, ZBTB7A, has no influence on the normal function of nucleated red blood cells after knockout, and expands the treatment means of thalassemia; the patient autologous hematopoietic stem cells are used as transplants, so that transplant immune rejection is completely avoided, and the risk of stem cell therapy is reduced.

Description

Hematopoietic stem cell and preparation method and application thereof

Technical Field

The invention relates to a hematopoietic stem cell, in particular to a hematopoietic stem cell with ZBTB7A gene unexpressed or reduced expression level, a preparation method and application thereof.

Background

The global haemoglobinopathy epidemiological report by WHO in 2008 indicates that haemoglobinopathy is a major health problem in approximately 71% of the population in 229 countries. Newborns born in these countries account for 89% of the worldwide newborns, with over 330000 newborns per year being associated with hemoglobinopathy (of which sickle cell disease is 83% and thalassemia 17%). The death from hemoglobinopathy is about 3.4% in children died under 5 years old.

In our country, there are about 56000 cases of severe β -thalassemia annually, of which at least 30000 require normative transfusions to survive, and about 5500 cases of severe α -thalassemia annually die of peri-natal death. In the southern China, the incidence of thalassemia gene defects reported in various places is 2.5% -20%, the incidence of thalassemia gene defects in the Guangdong province and the Guangxi province is respectively as high as 10% and 20%, and the number of thalassemia cases in the two provinces accounts for more than 2/5% of the total number of the China. Estimated as an average 3% gene-load rate of beta thalassemia, there are about 500 cases of severe beta thalassemia newborns born in Guangdong province each year, and about 5000 cases in total in 10 years. Therefore, thalassemia has become a social public health problem in high-incidence areas such as Guangdong, Guangxi, and the like. The areas with high incidence of thalassemia in China also have provinces and cities such as Hainan, Guizhou, Yunnan, Sichuan, Chongqing, Fujian, Hunan, Hubei, Jiangxi and the like. As the screening of the thalassemia is not brought into routine examination items before marriage and pregnant women in many provinces, basic medical staff have low cognition level on the thalassemia, and a forced marriage examination system is cancelled in recent years, so that the birth defect rate of various types of infants is obviously increased, and a plurality of infants with severe thalassemia are born every year. Taking Shenzhen city children hospital as an example, 12 cases of beta thalassemia major are newly diagnosed in 2006, and 9 cases in 2007.

Thalassemia, a hereditary chronic anemia caused by globin gene deficiency, is more common as beta and alpha thalassemia. At least 81 gene mutations (46 point mutations, 35 deletion mutations) of alpha-thalassemia have been found; the beta thalassemia gene has at least 186 mutation types, mainly point mutation.

Sickle cell anemia is an autosomal recessive hereditary disease. The cases are found mainly in African blacks, and also in mediterranean coastal countries such as the middle east, Greece, Italy, etc., and Indian, etc., and people who have a long time marriage with the above-mentioned regions and nations, and also in southern areas of China. The heterozygote state accounts for 20% of african blacks, 8% of american blacks, and 1/4 children are homozygotes in the marriage between heterozygotes, which leads to sickle-type anemia.

Sickle-type anemia patients have sickle-shaped hemoglobin (HbS) instead of normal Hb (HbA) because the glutamic acid at the 6 th amino acid of a beta-peptide chain is replaced by valine. When the partial pressure of oxygen decreases, HbS intermolecular interactions become helical multimers with very low solubility, causing the red blood cells to twist into sickle cells (sickling). The clinical manifestations are chronic hemolytic anemia, susceptibility to infection and recurrent pain crisis, so that chronic ischemia causes organ and tissue damage. The disease seriously damages the health of mothers and children, and can lead the death rate of fetuses to reach 5 percent and the death rate of pregnant women to reach 4.62 percent.

The current treatment of thalassemia and sickle cell anemia is mainly achieved by the following routes: 1, normative long-term blood transfusion and iron-removing treatment; hematopoietic stem cell transplantation (typically allotransplantation); 3, splenectomy, etc. Hematopoietic stem cell transplantation is the only treatment technology capable of radically treating thalassemia and sickle anemia at present, and mainly takes bone marrow hematopoietic stem cell allotransplantation as a main technology. However, bone marrow allografting is difficult to be applied clinically on a large scale because matched donors are rare and immune rejection is inevitable even if matching is successful.

The cord blood is proved to be rich in primitive hematopoietic cells, has strong proliferation potential, can overcome the obstacle of incomplete matching of human leukocyte antigens, and has lower incidence and degree of graft-versus-host disease and low mortality rate related to transplantation compared with bone marrow transplantation; the matching of cord blood is simpler than that of bone marrow, patients have more chances or receive transplantation treatment earlier, the blood transfusion frequency is reduced, on one hand, the treatment cost is reduced, and on the other hand, organ damage caused by iron overload is reduced. The sibling cord blood transplantation can completely cure severe thalassemia and sickle anemia. More importantly, the success rate of allogeneic or non-syngeneic cord blood stem cell transplantation is relatively high, so that more patients with thalassemia and sickle anemia without proper bone marrow donors can obtain the transplantation treatment opportunity, and a plurality of allogeneic or non-syngeneic cord blood transplants are reported to successfully treat thalassemia and sickle anemia. However, allogenic or non-syngenic cord blood transplantation still faces the problem of matching, and particularly under the condition that the public cord blood bank in China is incomplete, the large-scale popularization of allogenic cord blood for treating thalassemia or sickle-type anemia in clinic is still difficult.

While autologous hematopoietic stem cell transplantation can certainly solve the above problems of matching and immune rejection, the patient's own hematopoietic stem cells also have genetic defects and cannot be used directly for therapy. However, the method corrects the defective genes of the autologous stem cells or edits the related genes by a genetic engineering method in vitro, and then transplants the engineered stem cells back into the body of the patient, thereby not only repairing the inherent gene defects of the patient or changing the expression regulation of the related genes, but also avoiding the safety risk of immunological rejection, and being the most ideal, safe and effective treatment mode. More importantly, the transplantation of genetically engineered autologous hematopoietic stem cells in vitro can recover the lives of numerous patients with thalassemia and sickle anemia that have passed on in endless waiting for typing, so that typing is no longer a key problem for hindering the treatment of thalassemia and sickle anemia.

ZBTB7A gene, consisting of 584 amino acids. Is a ZBTB transcription factor, which is mainly combined with the DNA of the fetal hemoglobin gene through a C-terminal C2H 2-zinc finger structure and combined with a transcription inhibitor through an N-terminal BTB structure to form a compound, thereby inhibiting the transcription of the fetal hemoglobin gene. Studies have shown that when ZBTB7A gene is knocked out, fetal hemoglobin expression can be significantly increased.

In recent years, gene editing technologies have been developed in a breakthrough manner, and currently, ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases) and CRISPR (regularly clustered interspaced short palindromic repeats) -Cas9 are relatively mature gene editing technologies. The CRISPR-Cas9 is called as a third-generation gene editing technology, and compared with ZFNs and TALENs, the CRISPR-Cas9 is simple in construction, high in mutation efficiency and low in use cost. CRISPR systems each comprise the following parts (as shown in figure 1): 1) a PAM site, located downstream of the target sequence, of only a few nt (e.g., spCAS 9/NGG; saCAS9/NNGRRT), selecting a target sequence according to the site; 2) a sequence that recognizes and binds to a cleavage site of an RNA (gRNA or sgRNA) that recognizes a target sequence, generally about 20 nt; 3) tracrRNA, a palindromic RNA sequence following the target sequence; 4) cas9 endonuclease with independent nuclease activity, Cas9 contains 2 unique active sites, which are amino-terminal RuvC and HNH in the middle of the protein. The HNH active site in Cas9 cleaves the complementary DNA strand of the gRNA, and the RuvC active site cleaves the non-complementary strand. The working principle is as follows: the gRNA, tracrRNA and Cas9 form a complex that recognizes and binds to sequences complementary to the gRNA, then unzips the DNA double strand, forming an R-loop, hybridizing the gRNA to the complementary strand, leaving the other strand in a free single-stranded state, then cleaves the complementary DNA strand of the gRNA by the HNH active site in Cas9, cleaves the non-complementary strand by the RuvC active site, and finally introduces a DNA Double Strand Break (DSB).

In the prior art, there are currently several main approaches to the treatment of thalassemia and sickle cell anemia:

1) conservative treatment: normative long-term blood transfusion and iron-removing treatment, splenectomy and the like.

2) Eradication treatment: hematopoietic stem cell transplantation, mainly refers to allogeneic hematopoietic stem cell transplantation, such as bone marrow hematopoietic stem cells and umbilical cord blood hematopoietic stem cells.

The prior art has the following disadvantages:

1) conservative treatment (normative long-term blood transfusion, deferentization treatment, splenectomy, etc.) drawbacks: has great side effect and high cost, and can not be cured radically.

2) Eradication therapy (hematopoietic stem cell transplantation) drawbacks: allogenic hematopoietic stem cell transplantation presupposes proper mating. The success rate of bone marrow allotype is very low, and the immune rejection is relatively high. Although cord blood matches relatively easily, the public blood pool is incomplete, and the probability of finding cord blood with matching success is low.

However, autologous hematopoietic stem cell transplantation can completely solve the problems of matching and immune rejection, and has wider clinical application value. However, autologous hematopoietic stem cells of patients are gene-deficient and cannot be used directly for clinical treatment. The treatment can be achieved only by means of genetic engineering after the genome of the autologous hematopoietic stem cells is modified in vitro (such as knocking out or inhibiting the expression of ZBTB7A gene), and then the modified autologous hematopoietic stem cells are returned to the patient.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a hematopoietic stem cell with reduced or no expression of ZBTB7A gene, a preparation method and application of the hematopoietic stem cell.

In order to realize the purpose, the technical scheme is as follows: a hematopoietic stem cell which has a reduced or absent expression of ZBTB7A gene.

Preferably, the hematopoietic stem cells have disrupted ZBTB7A gene expression or a ZBTB7A gene knockout.

Preferably, the hematopoietic stem cells include at least one of cord blood hematopoietic stem cells, bone marrow hematopoietic stem cells and peripheral blood hematopoietic stem cells.

The invention provides the preparation method of the hematopoietic stem cell, which is obtained by interfering the expression of a hematopoietic stem cell ZBTB7A gene or knocking out the ZBTB7A gene of the hematopoietic stem cell.

Preferably, the ZBTB7A gene of the hematopoietic stem cell is knocked out by adopting a gene editing system or an expression vector for expressing the gene editing system, wherein the gene editing system comprises at least one of zinc finger nuclease, TALE nuclease and CRISPR-Cas9 system.

Preferably, the CRISPR-Cas9 system comprises at least one of a CRISPR-Sp Cas9 system, a CRISPR-Sp Cas9nickase system, a CRISPR-Sp dead Cas9-Fok1 system, a CRISPR-Sa Cas9 system, a CRISPR-Sa Cas9nickase system, a CRISPR-Sa dead Cas9-Fok1 system, and a CRISPR-Nm Cas9 system.

Preferably, the CRISPR-Sp Cas9 system includes a gRNA whose target sequence includes at least one of SEQ ID NO 1 to SEQ ID NO 38 and an Sp Cas9 protein.

Preferably, the target sequence of the gRNA includes at least one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38.

Preferably, the CRISPR-Sa Cas9 system includes a gRNA whose target sequence includes at least one of SEQ ID NOs 44 to 69 and a SaCas9 protein.

Preferably, the target sequence of the gRNA includes at least one of SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 59, SEQ ID NO 60, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 69.

Preferably, the CRISPR-Sp Cas9 system includes a gRNA composition consisting of a gRNA having a target sequence as set forth in SEQ ID NO:26 and a gRNA having a target sequence as set forth in SEQ ID NO:37, and an Sp Cas9 protein.

Preferably, the CRISPR-Sa Cas9 system includes a gRNA composition consisting of a gRNA whose target sequence is set forth in SEQ ID NO:48 and a gRNA whose target sequence is set forth in SEQ ID NO:63, and a SaCas9 protein.

Preferably, the CRISPR-Sp Cas9nickase system comprises a gRNA composition and a SpCas9nickase protein, wherein the gRNA composition consists of a gRNA with a target sequence shown in SEQ ID NO. 2 and a gRNA with a target sequence shown in SEQ ID NO. 26, or consists of a gRNA with a target sequence shown in SEQ ID NO. 2 and a gRNA with a target sequence shown in SEQ ID NO. 28, or consists of a gRNA with a target sequence shown in SEQ ID NO. 11 and a gRNA with a target sequence shown in SEQ ID NO. 26, or consists of a gRNA with a target sequence shown in SEQ ID NO. 11 and a gRNA with a target sequence shown in SEQ ID NO. 28, or consists of a gRNA with a target sequence shown in SEQ ID NO. 11 and a gRNA with a target sequence shown in SEQ ID NO. 32.

The invention provides an application of ZBTB7A gene as a target spot in preparing or screening medicaments for treating thalassemia or sickle type anemia.

The invention provides application of the hematopoietic stem cells in preparing a medicament for treating thalassemia or sickle-type anemia.

Has the advantages that:

1. the fetal hemoglobin gene inhibitor ZBTB7A is knocked out or interfered, so that the expression of the fetal hemoglobin can be improved after the fetal hemoglobin gene inhibitor ZBTB7A is not expressed or the expression level is reduced, and the treatment method of thalassemia and sickle-type anemia is expanded.

2. The autologous hematopoietic stem cells of patients are used for knocking out or interfering with expression of fetal hemoglobin gene inhibitor ZBTB7A to prepare the engineered autologous hematopoietic stem cells for treating thalassemia and sickle anemia, so that the transplantation immune rejection is completely avoided, and the treatment risk of stem cells is reduced.

3. The fetal hemoglobin gene inhibitor is knocked out by using the CRISPR-Cas9 technology, so that the operation is simple and convenient, the efficiency is high, and the time consumption is short.

Drawings

FIG. 1 is a plasmid map of a commercial CRISPR-Sp Cas9 vector pX 458;

FIG. 2 is a diagram of a gRNA screening T7 endonuclease 1 enzyme digestion detection electrophoresis of CRISPR-Sp Cas9 in example 1 of the present invention;

FIG. 3 is a plasmid map of a commercial CRISPR-Sa Cas9 vector pX 601;

fig. 4 is an electrophoresis diagram of enzyme digestion detection of gRNA screening T7 endonuclease 1 of CRISPR-Sa Cas9 in embodiment 2 of the present invention;

fig. 5 is an electrophoresis diagram of the T7 endonuclease 1 enzyme digestion detection of the combination of CRISPR-Sp Cas9 and CRISPR-Sa Cas9 respectively with grnas in embodiment 3 of the present invention;

FIG. 6 is a plasmid map of a commercial CRISPR-Sp Cas9nickase vector pX 461;

FIG. 7 is an electrophoresis diagram of enzyme cleavage detection of T7 endonuclease 1 by gRNA combined screening of CRISPR-Sp Cas9nickase described in example 4 of the present invention.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

The experimental materials used in the following examples include: a commercial CRISPR-Sp Cas9 vector, such as pX458 (map shown in figure 1); a commercial CRISPR-Sa Cas9 vector, such as pX601 (map shown in fig. 3); a commercial CRISPR-Sp Cas9nickase vector, as pX461 (map see fig. 6); HEK293T cells; coli competent cell TOP 10.

Example 1

Knocking out a ZBTB7A gene by using a CRISPR-Sp Cas9 gene editing system, and specifically comprising the following steps:

1. gRNA preparation

(1) Designing a 20nt gRNA sequence according to the sequence of ZBTB7A gene, wherein the target sequence of the gRNA is shown as one of SEQ ID NO 1-SEQ ID NO 38;

(2) separately synthesizing a sense strand and an antisense strand of the target sequence (the 5 '-end of the sense strand plus cacc, if the first nucleotide at the 5' -end of the sense strand is not guanine G, the 5 '-end of the sense strand plus caccG; the 5' -end of the antisense strand plus aaac, if the first nucleotide at the 5 '-end of the sense strand is not guanine G, the 3' -end of the antisense strand plus C);

(3) the sense strand and the antisense strand synthesized as described above are mixed, treated at 90 ℃ and then naturally cooled to room temperature to carry out annealing treatment, thereby synthesizing a double-stranded DNA fragment having a cohesive end.

The sense strand of the target sequence of the gRNA of CRISPR-Sp Cas9 system designed against the ZBTB7A gene is as follows:

2. preparation of the vector

(1) Amplifying and extracting pX458 plasmid, and determining the concentration of the plasmid;

(2) the restriction enzyme Bbs I is adopted to cut pX458, and loading buffer is added to terminate the reaction after the enzyme is cut for 1h at 37 ℃.

(3) After agarose gel electrophoresis, cutting gel to recover linearized plasmid pX458, and determining the concentration of the recovered product, and storing at-20 ℃ for later use.

3. Ligation transformation

(1) Carrying out ligation reaction on the linearized pX458 vector recovered from the gel cutting and the annealed double-stranded DNA fragment;

(2) and (3) transforming the escherichia coli competent cell TOP10 by a ligation product heat shock method, adding a sterile LB liquid culture medium (without antibiotics) into each centrifuge tube after transformation, uniformly mixing, and placing in a constant temperature shaking table at 37 ℃ and 200rpm for shaking culture for 45min to recover the thalli.

(3) Recovered TOP10 cells were plated on LB solid plates (Amp)⁺) And inversely placing the mixture in a constant temperature incubator at 37 ℃ for static culture for 12-16 h.

(4) Single colonies were picked from the above plates and inoculated into LB liquid medium (Amp)⁺) Medium-scale culture.

(5) The above-mentioned bacterial solutions were sequenced using a primer SeqF (5'-ATTTTTGTGATGCTCGTCAG-3') (SEQ ID NO:39), respectively;

(6) extracting plasmids from the bacterial liquid with correct sequencing, measuring the concentration of the plasmids, and storing at-20 ℃ for later use.

4. Cell transfection

(1) HEK293T cell plating;

(2) the plasmids extracted in 3(6) of example 1 were transfected into HEK293T cells respectively by using a Lipofectamine 3000 kit;

(3) the transfected cells were cultured for 48 hours and harvested by centrifugation.

5. T7E1 enzyme digestion analysis mutation efficiency

(1) Extracting cell genome from the cells collected in the step 4(3) and detecting the genome concentration;

(2) PCR primers were designed upstream and downstream of the gRNA binding site (target sequence), respectively, as shown in table 1;

TABLE 1T7E1 list of primers for enzyme digestion analysis

(3) Amplifying target fragments with target sites by using a PCR method respectively;

(4) purifying and recovering PCR products, and determining the product concentration;

(5) annealing the purified PCR product, namely heating to 95 ℃, preserving heat for 10min, and then cooling to room temperature at the speed of reducing the temperature by 2-3 ℃ every 30 s;

(6) t7 endonuclease 1(T7E1) was added to each tube of the annealed product, and the resulting mixture was digested at 37 ℃ for 1 hour in mock groups (untransformed cells).

(7) The cleavage effect was checked by 2% agarose gel electrophoresis (see FIG. 2).

The results are shown in fig. 2, (a) (b) shows the detection result of T1 region, and (c) shows the detection result of T2 region, each lane is named by the number of the corresponding gRNA target sequence, wherein the underlined lane is a fragment with relatively high cleavage efficiency of T7E1, and the cleavage efficiency of the gRNA corresponding to the lane to the ZBTB7A gene is demonstrated to be high according to the detection principle of T7E 1. The gRNA target sequences with high cutting efficiency on the ZBTB7A gene are respectively SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 36, SEQ ID NO. 37, and SEQ ID NO. 38.

6. Sanger sequencing method for analyzing mutation efficiency

(1) Randomly selecting one gRNA from the selected gRNAs with high cutting effect on the ZBTB7A gene, and further analyzing the mutation efficiency of the selected gRNA by adopting a Sanger sequencing method, wherein the gRNA corresponding to a target sequence SEQ ID NO. 26 is taken as an example;

(2) the purified PCR product obtained in (4) of example 5 was subjected to PCR Mix with a century Master PCR Mix to which an adenine (A) was added at the 3' -end: mixing 2 μ g of the PCR product in 2.1 with Master PCR Mix at a ratio of 1:1(V: V), and reacting at 72 deg.C for 30 min;

(3) cutting gel after electrophoresis with 1% agarose gel to recover the target fragment and determining the concentration of the recovered product;

(4) with TaKaRa pMD^TMLigation with 18-T Vector Cloning Kit: preparing a reaction system according to the following table 2, and reacting for 30min at 16 ℃;

TABLE 2 ligation of pMD^TMReaction system of 18-T carrier

ddH₂O	Make up to 10 mu L
		pMD18-T Vector(5×)	1μL(10ng)
Segment of interest	0.1pmol～0.3pmol
		Solution I	5μL

(5) Transforming escherichia coli competent cells TOP10 by the ligation product hot shock method, adding sterile LB liquid culture medium (without antibiotics) into each centrifuge tube after transformation, uniformly mixing, placing in a constant temperature shaking table at 37 ℃ and 200rpm, and performing shaking culture for 45min to recover thalli;

(6) recovered TOP10 cells were plated on LB solid plates (Amp)⁺) Inversely placing the mixture in a constant temperature incubator at 37 ℃ for static culture for 12-16 h;

(7) single colonies growing on the plates were randomly picked and subjected to sanger sequencing with the following results:

a total of 35 randomly selected single colonies were sequenced, of which 15 in total were null-sequencing with self-ligation of the T vector, 9 with mutation in the sequence, 11 with no mutation in the sequence, and mutation efficiency was 45%.

Example 2

Knocking out a ZBTB7A gene by using a CRISPR-Sa Cas9 gene editing system, and specifically comprising the following steps:

1. gRNA preparation

(1) Designing a gRNA sequence of 21nt according to the sequence of ZBTB7A gene, wherein the target sequence of the gRNA is shown in one of SEQ ID NO:44-SEQ ID NO: 38;

The sense strand of the target sequence of the gRNA of CRISPR-Sa Cas9 system designed against the ZBTB7A gene is as follows:

2. preparation of the vector

(1) Amplifying and extracting pX601 plasmid, and determining the concentration of the plasmid;

(2) the pX601 is cut by restriction enzyme Bsa I, and the reaction is terminated by adding loading buffer after 1h of enzyme cutting at 37 ℃.

(3) After agarose gel electrophoresis, cutting gel and recovering linearized plasmid pX601, and determining the concentration of the recovered product, and storing at-20 ℃ for later use.

3. Ligation transformation

(1) Performing a ligation reaction on the linearized pX601 vector recovered from the gel cutting and the annealed double-stranded DNA fragment;

(5) The above-mentioned bacterial solutions were sequenced using primer 601SaF (5'-TTCCTTGACCCTGGAAGGTG-3') (SEQ ID NO:70), respectively;

4. Cell transfection

(1) HEK293T cell plating;

(2) the plasmids extracted in 3(6) of example 2 were transfected into HEK293T cells respectively by using a Lipofectamine 3000 kit;

5. T7E1 enzyme digestion analysis mutation efficiency

(2) PCR primers were designed upstream and downstream of the gRNA binding site (target sequence), respectively, as shown in table 3;

TABLE 3 List of primers for enzyme digestion analysis of T7E1

(6) t7 endonuclease 1(T7E1) was added to each tube of the annealed product, and mock (untransformed cells) and blank Ck (ddH without T7E1) were set₂O is substituted) and enzyme digestion is carried out for 1h at 37 ℃;

(7) the cleavage effect was checked by 2% agarose gel electrophoresis (see FIG. 4).

The results are shown in FIG. 4, where (a) is the result of detection of the region T1, (b) (c) is the result of detection of the region T2, and (d) (e) is the result of detection of the region T3. Each lane is named by the number of the corresponding gRNA target sequence, wherein the underlined lane is a fragment with relatively high T7E1 enzyme digestion efficiency, and the detection principle of T7E1 shows that the gRNA corresponding to the lane has relatively high cleavage efficiency on the ZBTB7A gene. The gRNA target sequences with higher cutting efficiency on the ZBTB7A gene are respectively SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65 and SEQ ID NO. 69.

6. Sanger sequencing method for analyzing mutation efficiency

(1) Randomly selecting one gRNA from the selected gRNAs with high cutting effect on the ZBTB7A gene, and further analyzing the mutation efficiency of the selected gRNA by adopting a Sanger sequencing method, wherein the gRNA corresponding to a target sequence SEQ ID NO. 48 is taken as an example;

(2) the purified PCR product obtained in example 5 or (4) was added with adenine (A) at the 3' -end of the PCR product using the Kangji Master PCR Mix: mixing 2 μ g of the PCR product in 2.1 with Master PCR Mix at a ratio of 1:1(V: V), and reacting at 72 deg.C for 30 min;

(4) with TaKaRa pMD^TMPerforming a ligation reaction with 18-T Vector Cloning Kit, wherein the reaction system is shown in Table 2 and reacts for 30min at 16 ℃;

a total of 35 randomly selected single colonies were sequenced, with a total of 10 null-sequencing with self-ligation of the T-vector, 9 with mutated sequences, 16 without mutated sequences, and a mutation efficiency of 36%.

Example 3

The CRISPR-Sp Cas9 and CRISPR-Sa Cas9 gene editing systems are adopted respectively to knock out the ZBTB7A gene in combination with double gRNA combinations, and the specific implementation steps are as follows:

1. HEK293T cells were co-transformed with pX458 vector prepared in 3(6) of example 1 and connected with SEQ ID NO 26 and SEQ ID NO 37 by the method of 4 of example 1, and then subjected to T7E1 enzyme digestion according to the step of 5 to analyze the cleavage efficiency;

2. HEK293T cells were co-transformed with pX601 vectors prepared in 3(6) of example 2 and connected with SEQ ID NO 48 and SEQ ID NO 63 by the method of 4 of example 2, and then subjected to T7E1 enzyme digestion according to the step of 5 to analyze the cleavage efficiency;

3. the above digestion effect was detected by 2% agarose gel electrophoresis, and the results are shown in FIG. 5: each lane is named with the type of CRISPR system (Sp Cas9 and Sa Cas9) and target sequence number of the gRNA combination, corresponding to the detection results of 1 and 2 in this example, respectively, from left to right. According to the detection principle of T7E1, the higher enzyme digestion efficiency indicates the higher mutation efficiency. Therefore, as shown in the figure, the combination of the gRNAs with the target sequences of SEQ ID NO. 26 and SEQ ID NO. 37 collocated with Sp Cas9 and the combination of the gRNAs with the target sequences of SEQ ID NO. 48 and SEQ ID NO. 63 collocated with Sa Cas9 have higher mutation efficiency on the ZBTB7A gene;

4. mutation efficiency was analyzed by Sanger sequencing according to the procedure of example 1 or 6 in example 2: (1) sp Cas9 is matched with a gRNA combination with target sequences of SEQ ID NO 26 and SEQ ID NO 37 to sequence 55 randomly selected single colonies, wherein the number of the single colonies is 5 in total, the number of the single colonies is 36 in which T vector self-connection occurs, the number of the single colonies is 14 in which the sequence is not mutated, and the mutation efficiency is 72%; (2) the Sa Cas9 matched with the gRNA combination with target sequences of SEQ ID NO 48 and SEQ ID NO 63 sequenced a total of 26 randomly selected single colonies, wherein the number of invalid sequences in which the T vector self-ligation occurs is 0, the number of sequences in which mutations occur is 14, the number of sequences in which mutations do not occur is 12, and the mutation efficiency is 53.8%.

Example 4

The CRISPR-Sp Cas9nickase gene editing system is adopted to knock out the ZBTB7A gene in combination with double gRNA, and the specific implementation steps are as follows:

1. preparation of gRNA combinations

(1) Selecting gRNAs with higher mutation efficiency analyzed by T7E1 in example 1, selecting matched gRNAs within 200bp of the DNA complementary strand of the combined target sequence, and the matching combination is shown in Table 4;

TABLE 4gRNA pairing combinations

(2) Fragments necessary for the above-described pairing are selected from the sense strand and the antisense strand of the gRNA synthesized in 1(2) of example 1, and double-stranded DNA fragments having sticky ends are annealed according to the method of 1(3) of example 1.

2. Preparation of the vector

(1) Amplifying and extracting pX461 plasmid, and determining the concentration of the plasmid;

(2) the pX461 is cut by restriction enzyme Bbs I, and the reaction is terminated by adding loading buffer after 1h of enzyme cutting at 37 ℃.

(3) The linearized plasmid pX461 was recovered by agarose gel electrophoresis and the concentration of the recovered product was determined and stored at-20 ℃ for further use.

3. Ligation transformation

(1) Performing a connection reaction on the linearized pX461 carrier recovered from the gel cutting and the annealed gRNA double strand;

(2) transforming escherichia coli competent cells TOP10 by a heat shock method, adding sterile LB liquid culture medium (without antibiotics) into each centrifuge tube after transformation, uniformly mixing, placing in a constant temperature shaking table at 37 ℃ and 200rpm, and carrying out shaking culture for 45min to recover thalli.

(4) Single colonies were picked from the above plates and inoculated into LB liquid medium (Amp +) for scale-up culture.

(5) Sequencing the bacterial solutions by primers SEQ ID NO. 39 respectively;

4. Cell transfection

(1) HEK293T cell plating;

(2) co-transfecting HEK293T cells with plasmids extracted in 3(6) by using a Lipofectamine 3000 kit according to a combination mode shown in a table 4;

(3) the transfected cells were cultured for 48 hours, and the cells were collected.

5. T7E1 enzyme digestion analysis mutation efficiency

(2) PCR amplification of a target fragment with a target site was performed using the T1 region primers SEQ ID NO:40 and SEQ ID NO:41 shown in Table 1;

(3) purifying and recovering PCR products, and determining the product concentration;

(4) annealing the purified PCR product, namely heating to 95 ℃, preserving heat for 10min, and then cooling to room temperature at the speed of reducing the temperature by 2-3 ℃ every 30 s;

(5) t7 endonuclease 1(T7E1) was added to each tube of the annealed product, and mock (untransformed cells) and blank (ddH without T7E1) were set₂O), digesting for 1h at 37 ℃.

(6) The cleavage effect was detected by 2% agarose gel electrophoresis, and the results are shown in FIG. 7: the electrophoresis channel is named by the target sequence number of each gRNA combination used in the experiment, and the band () represents the fragment of the sample without the T7E1 enzyme, i.e., the blank control group, and the underlined lane is the fragment with higher enzyme digestion efficiency of T7E 1. Combining with the detection principle of T7E1, the diagram shows that the CRISPR-Sp Cas9nickase system matches a gRNA combination with target sequences of SEQ ID NO. 2 and SEQ ID NO. 26, a gRNA combination with target sequences of SEQ ID NO. 2 and SEQ ID NO. 28, a gRNA combination with target sequences of SEQ ID NO. 11 and SEQ ID NO. 26, a gRNA combination with target sequences of SEQ ID NO. 11 and SEQ ID NO. 28, and a gRNA combination with target sequences of SEQ ID NO. 11 and SEQ ID NO. 32, and has high mutation efficiency on ZBTB7A genes, wherein the gRNA combination with target sequences of SEQ ID NO. 11 and SEQ ID NO. 26 has optimal mutation efficiency.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> Guangdong Chimeng medical science & technology Limited

<120> hematopoietic stem cell, preparation method and application thereof

<160> 76

<170> PatentIn version 3.3

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ctctccttcc ccactgctgc 20

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ttccccactg ctgcaggtct 20

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cactgctgca ggtctcggcg 20

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gcaggtctcg gcgcggaaga 20

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aagatggccg gcggcgtgga 20

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ggcggcgtgg acggccccat 20

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cacagcagcg acatcctgag 20

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acagcagcga catcctgagt 20

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tgagtgggct gaacgagcag 20

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gctgtgcgac gtggtgatcc 20

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gtgcgacgtg gtgatcctgg 20

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cgacgtggtg atcctggtgg 20

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cgagttcccc acgcaccgct 20

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ccccacgcac cgctcggtgc 20

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tacttcagcg gcgcccacga 20

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ccacgacggc gacgtctacc 20

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ctacccggcc tggtcgcaga 20

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ggagaagaag atccgagcca 20

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gaagtgcccc atctgcgaga 20

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catctgcgag aaggtcatcc 20

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gagaaggtca tccagggcgc 20

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gcgacacatc cgcacccaca 20

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ctacgagtgc aacatctgca 20

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tctgcaaggt ccgcttcacc 20

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gatggggccg tccacgccgc 20

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cgctgctgtg gtcggggaac 20

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caggatgtcg ctgctgtggt 20

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ggatcaccac gtcgcacagc 20

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ggaactcgcg gccctccacc 20

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agcggtgcgt ggggaactcg 20

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ggccagcacc gagcggtgcg 20

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tgcaggcggc cagcaccgag 20

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ccgggtagac gtcgccgtcg 20

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cttctccacc ttctgcgacc 20

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acttctggaa ggccttggct 20

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gatgtgtcgc ggcagcttgc 20

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gtgtgggtgc ggatgtgtcg 20

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cttgcagatg ttgcactcgt 20

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atttttgtga tgctcgtcag 20

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ctggccaaca tggtgatagc 20

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gggttgctct ggaagaactc 20

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ctgcagcaga tgatgtcatc ggtg 24

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gtgtctgtgg gtgcatgttt gcatg 25

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cggcggcgtg gacggcccca t 21

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gtcgctgctg tggtcgggga a 21

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cccgaccaca gcagcgacat c 21

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ccgctgctcg ttcagcccac t 21

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tggtgatcct ggtggagggc c 21

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ggggaactcg cggccctcca c 21

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ctcaccgtca gcacagccaa c 21

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ctccagcagg cgggcggcgc t 21

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ggcgcacacg tggctcacgg c 21

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gtcggcgccc gcgtcggccg c 21

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agcgcaacct cctccgcgcc a 21

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tcctccgcgc caaggagtac c 21

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ccgcgggggg caggctgttc a 21

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tttggggcgt ccgatgatga c 21

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ggggacgagg gcgacagcaa c 21

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aacccgggtc tgtggccaga g 21

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catcccgctc tggccacaga c 21

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cgggatgagg acgcccccac c 21

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ccgctcccga caggaagccc g 21

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ccgcggcggg ggacagcgac g 21

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tctccacctt ctgcgaccag g 21

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gcacttctgg aaggccttgg c 21

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tcgcggcagc ttgccggcgc c 21

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gggcttctcg cccgtgtggg t 21

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ctcgtagggc ttctcgcccg t 21

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cccacacggg cgagaagccc t 21

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ttccttgacc ctggaaggtg 20

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ctggccaaca tggtgatagc ctgtctct 28

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gggttgctct ggaagaactc gaggtactc 29

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accgcgctca tggacttcgc ctaca 25

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gtagtagtcc atgacgccct tgtcgtcg 28

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gcgactgcaa cggcttagac ttctatgg 28

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tctgtgcgtg tacgtgtctg tgtgcac 27

Claims

1. A method for preparing hematopoietic stem cells with reduced or no expression of ZBTB7A gene, which comprises interfering the expression of the hematopoietic stem cell ZBTB7A gene or knocking out the ZBTB7A gene of the hematopoietic stem cell to obtain the hematopoietic stem cell;

wherein the interference or knockout adopts a gene editing system CRISPR-Sp Cas9 system;

the CRISPR-Sp Cas9 system includes a gRNA composition consisting of a gRNA having a target sequence shown in SEQ ID NO:26 and a gRNA having a target sequence shown in SEQ ID NO:37, and an Sp Cas9 protein.

2. The method for producing hematopoietic stem cells according to claim 1, wherein the hematopoietic stem cells include at least one of cord blood hematopoietic stem cells, bone marrow hematopoietic stem cells and peripheral blood hematopoietic stem cells.