CN112111528B - Repair method for abnormal splicing of introns - Google Patents
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Abstract
The invention discloses a repair method for abnormal splicing of introns, which is a technology for targeting and knocking out abnormal mutation sites IVS2-654C > T in beta-thalassemia (thalassemia) by using a CRISPR-Cas9 gene editing technology, and is characterized in that a guide RNA sequence (sgRNA) capable of recognizing and guiding Cas9 protein to a target gene target sequence is designed and synthesized, and the guide RNA sequence and Cas9 protein mixture are electrically transduced into beta-thalassemia IVS2-654C > T hematopoietic stem cells, so that the abnormal splicing mutation sites are efficiently destroyed, and normal shearing and expression of beta-globin genes are recovered. The invention can edit blood transfusion dependent beta-thalassemia IVS2-654C > T by using the existing gene editing technology, has high editing efficiency, and can reconstruct the blood system of a patient and treat thalassemia after autologous transplantation of edited hematopoietic stem cells of the patient.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for repairing an abnormal splicing site of an intron.
Background
In recent years, an adaptive immune mechanism for protecting against invasion of foreign DNA fragments such as phage and plasmids has been elucidated in bacteria and archaebacteria. The system consists of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (CAS) genes. The immune interference process of CRISPR systems mainly consists of 3 phases: adaptation, expression and interference. In the adaptation phase, the CRISPR system integrates short stretches of DNA from phage or plasmid between the leader sequence and the first repeat sequence, each integration accompanied by replication of the repeat sequence, thereby forming a new repeat-spacer unit. In the expression phase, the CRISPR locus will be transcribed into a segment of CRISPR RNA (crRNA) precursor (pre-crRNA) which in the presence of Cas protein and tracrRNA will be further processed into small crrnas at the repeat sequence. The mature crRNA forms a Cas/crRNA complex with the Cas protein. In the interference stage, the crRNA guides the Cas/crRNA complex to search a target through a region complementary to the target sequence, and double-strand DNA at the target position is broken by nuclease activity of the Cas protein at the target position, so that the target DNA loses the original function. Wherein the 3 bases immediately 3' to the target must be in the form of 5' -NGG-3' to constitute the PAM (protospacer adjacent motif) structure required for the Cas/crRNA complex to recognize the target.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems are divided into three families of type I, II, III, where the type II system requires only Cas9 protein to process pre-crRNA into mature crRNA that binds to tracrRNA with the aid of trans-encoded small RNA (tracrRNA). It was found that by artificial construction of the simulated crRNA: the single-chain chimera of the tracrRNA complex guides RNA (guide RNA), which can effectively mediate the recognition and cutting of the target spot by the Cas9 protein, thereby providing a broad prospect for modifying the target DNA in the target species by using the CRISPR system.
Beta-thalassemia is a common genetic disease that causes adult hemoglobin abnormalities due to beta-globin gene deficiency, with "thalassemia" gene carriers in our country being about 3000 tens of thousands, involving nearly 3000 tens of thousands of families, 1 million populations, of which heavy and intermediate "thalassemia" patients are about 30 tens of thousands. Among them, IVS2-654C > T genotype is a more common "poor land" in our country, and the pathogenesis is that the 654 th base in the 2 nd intron of HBB gene generates C > T mutation, thereby generating abnormal splice sites, resulting in an extra 73nt more exons in beta-globin mRNA and early termination of translation. At present, long-term blood transfusion and iron removal treatment are needed for intermediate type and heavy type patients to maintain life, the only radical cure mode is allogeneic hematopoietic stem cell transplantation fine implantation, but the main implementation obstacles are the shortage of blood resources in China, difficult allogeneic hematopoietic stem cell matching, transplantation related complications and the like. Among them, gene therapy using lentiviral vectors has great potential, but semi-random vector integration has an oncogenic risk. Meanwhile, the expression original in the slow virus is gradually silenced in the long-term homing and self-renewing processes of the hematopoietic stem cells, so that the curative effect is reduced, and the aim of curing for life cannot be fulfilled. In addition, high-concentration, high-quality lentiviruses required clinically are extremely high in requirements for equipment and technology, and therefore, cost is hardly reduced. Thus, parallel, safer, lower cost clinical protocols are highly desirable.
An ideal gene therapy method is to repair or destroy the traditional poor mutation in the DNA of hematopoietic stem cells of patients, restore the gene function, and permanently produce wild-type adult beta-globin under the action of endogenous transcription control factors, so as to normally differentiate into erythroid cells. The repair mode of the DNA sequence after gene editing is mainly Non-homologous end joining (Non-homologous end joining, NHEJ) repair, the proportion of homologous recombination mediated repair (Homology directed repair, HDR) is extremely low, and efficient HDR efficiency is required for repairing point mutation. The strategy of the invention aims at abnormal mutation of pathogenic sites and can destroy the mutation sites by only realizing NHEJ mutation through targeting and cutting target DNA is more feasible. Clinically, the purpose of curing can be achieved by transplanting autologous hematopoietic stem cells repaired in a NHEJ mode back into the body after gene editing.
Disclosure of Invention
The invention provides a repair method for abnormal splicing of introns caused by mutation of beta-globin gene (HBB) IVS 2-264C > T.
The invention utilizes CRISPR-Cas9 system to repair the abnormal splicing of introns caused by IVS 2-650C > T. When Cas9 targets the cleavage site, the 3 bases immediately adjacent to the 3' end of the target must be in the form of 5' -NGG-3' in the choice of available targeting sequences, thus constituting the PAM (protospacer adjacent motif) structure recognized by Cas9 itself. However, there is no suitable PAM targeted cleavage near the pathogenic mutation site to disrupt the aberrant splice site, which results in the inability to directly cleave and repair the IVS 2-504 c > t mutation site using the CRISPR-Cas9 system.
The inventors have conducted intensive studies on aberrant splicing of introns caused by IVS 2-264C > T and found that mutation of IVS 2-264C > T results in the production of an additional splice donor site "AAGGTAATA" in the second intron of HBB, resulting in the production of an aberrant beta-globin mRNA of 73 more bases, such that translation is prematurely terminated. Theoretically, disrupting the function of the extra splice donor site due to IVS 2-650C > T in the HBB gene region, so that it does not trigger abnormal variable cleavage, allows the genomic region to be transcribed to produce normal β -globin mRNA and translated to produce normal β -globin.
In one embodiment, the additional splice donor site "AAGGTAATA" due to IVS 2-254C > T can be disrupted by base insertion, deletion, alteration, frameshift mutation or knockout.
In one embodiment, the CRISPR-Cas9 system may be employed to disrupt the additional splice donor site "AAGGTAATA" due to IVS 2-254 c > t; preferably, when using the CRISPR-Cas9 system, the targeting sequence of the designed sgrnas is preferably in the range of 20bp upstream of the IVS 2-264 c > t site to 70bp downstream of the IVS 2-264 c > t site.
In the present invention, the CRISPR-Cas system refers to a nuclease system suitable for artificially engineered CRISPR-Cas systems, derived from archaebacteria type II (CRISPR) -CRISPR-associated protein (Cas) systems, which is simpler and more convenient to operate than ZFNs and TALENs.
The invention uses RNA-guided endonucleases (RNA-guided endonucleases, RGENs) to achieve specific cleavage of target gene sequences. RGENs consist of chimeric guide RNAs and Cas9 proteins, wherein the former is the fusion of CRISPR RNAs (crRNAs) in naturally occurring type II CRISPR-Cas systems with trans-activating crRNAs (tracrrRNAs) into a single strand of guide RNAs (sgRNAs) that bind to Cas9 proteins and direct the latter to specifically cleave the target DNA sequence, which will form a double strand break (double strand break, DSB), which is repaired by error-prone Non-homologous end joining (Non-homologous end joining, NHEJ) that is highly effective in causing a frameshift of the target gene, thereby effecting disruption of the site of pathogenic mutation.
The Cas9 may be selected from Streptococcus pyogenes, staphylococcus aureus or n.menningitidis-derived Cas9. The Cas9 may be selected from wild-type Cas9, or may be selected from mutant Cas9; the mutant Cas9 does not result in loss of cleavage activity and targeting activity of Cas9.
In other embodiments, other Cas enzymes may also be used in place of Cas9.
In the invention, because a targeting sequence suitable for Cas9 protein cutting is not arranged near the IVS 2-264C > T site, cutting is selected from the range of 20bp upstream of the IVS 2-264C > T site to 70bp downstream of the IVS 2-264C > T site, and the cutting can cause abnormal translation of an additional splice donor site 'AAGGTAATA' generated by the IVS 2-264C > T, thereby achieving the repairing effect. Therefore, in the invention, after the Cas9 cuts the target sequence, an exogenous DNA donor sequence is not required to be introduced for homologous recombination repair.
In one embodiment, cleavage of the target sequence is performed with sgRNA-1, which targets CAGTGATAATTTCTGGGTTA (SEQ ID No. 1), the sgRNA-1 can direct Cas9 to cleave DNA 6 bases upstream of the IVS 2-264C > T site, and cleavage alone results in base loss, such that additional splice donor sites due to IVS 2-264C > T result in additional base loss, disrupting the additional splice donor sites.
In another embodiment, sgRNA-1 can also be used in combination with sgRNA-2, which targets TAAATTGTAACTGATGTAAG (SEQ ID No. 2) for the sequence of sgRNA-2, which can guide Cas9 to cleave DNA 53 bases downstream of the IVS 2-504C > T site; the combination of sgRNA-1 and sgRNA-2 also disrupts additional splice donor sites due to IVS 2-264C > T.
Whether used with sgRNA-1 alone or with both sgRNA-1 and sgRNA-2, the function of the additional splice donor site can be disrupted so that it does not trigger aberrant variable cleavage, thereby allowing the genomic region to be transcribed to produce normal β -globin mRNA and translated to produce normal β -globin.
If the second intron region of normal human HBB is subjected to sgRNA1 or sgRNA1+2 targeted gene editing, and corresponding base loss or large fragment DNA deletion is generated, normal shearing or transcription of beta-globin mRNA is not affected. This means that when the allele causing the disease is edited, the expression of β -globin is not affected even if another normal allele is edited at the same time.
Detailed description of the invention:
in one aspect, the invention provides a method of repairing abnormal splicing of introns caused by mutation of an IVS 2-264C > T of HBB (β -globin gene) in a cell, said IVS 2-264C > T being capable of causing an additional splice donor site, said method comprising the step of gene editing of HBB using a CRISPR-Cas9 system, said gene editing of HBB using a CRISPR-Cas9 system to inactivate said additional splice donor site, said CRISPR-Cas9 system comprising Cas9 and at least one sgRNA targeting a target sequence.
Such inactivation includes disruption of the function of the additional splice donor site by base insertion, deletion, alteration, frameshift mutation, or knock-out.
Further, the targeting sequence of the sgRNA is 20bp upstream of the IVS 2-650C > T site to 70bp downstream of the IVS 2-650C > T site.
In one embodiment, the sgRNA comprises sgRNA-1; in other embodiments, the sgRNA comprises sgRNA-1 and sgRNA-2.
The targeting sequence of the sgRNA-1 is CAGTGATAATTTCTGGGTTA; the targeting sequence of sgRNA-2 is TAAATTGTAACTGATGTAAG.
Further, the sequence of the additional splice donor site is AAGGTAATA.
Further, the cells are hematopoietic stem cells, preferably, the cells are CD34 + Is selected from the group consisting of hematopoietic stem and progenitor cells; more preferably, the cell is an ex vivo cell.
In one embodiment, the sgRNA comprises a chemical modification of a base. In preferred embodiments, the sgrnas comprise chemical modifications of any one or any several bases from 1 st to n th base at the 5 'end, and/or chemical modifications of any one or any several bases from 1 st to n th base at the 3' end; the n is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, the sgrnas comprise chemical modifications of one, two, three, four or five bases at the 5 'end, and/or chemical modifications of one, two, three, four or five bases at the 3' end. For example, the 1 st, 2 nd, 3 rd, 4 th, 5 th or 1-2 nd, 1-3 rd, 1-4 th, 1-5 th base of the 5' end of the sgRNA is chemically modified; and/or, carrying out chemical modification on the 1 st base, the 2 nd base, the 3 rd base, the 4 th base, the 5 th base or the 1 st to 2 nd bases, the 1 st to 3 rd bases, the 1 st to 4 th bases and the 1 st to 5 th bases of the 3' end of the sgRNA. In a preferred embodiment, the chemical modification is one or any of methylation modification, fluorination modification or thio modification.
In a preferred embodiment, the complex comprising Cas9 and sgRNA is introduced into the cell using an electrotransformation method.
Further, the molar ratio of Cas9 to sgRNA is 1:1-3, preferably 1:2, more preferably 1:3.
Further, the Cas9 and sgrnas form a complex by incubation; preferably, the temperature of the incubation is 20-50 ℃, preferably 25-37 ℃; preferably, the incubation time is 2-30 minutes, preferably 5-20 minutes.
Further, the Cas9 and sgRNA containingThe ratio of complex to cell is 20-100 μg complex: (1X 10) 2 -1×10 6 Individual) cells, preferably 30 μg complex: (1X 10) 3 -1×10 5 Individual) cells.
Further, cells after electrotransformation were cultured on CD34 + Extracting genome DNA of the cells obtained in the steps for 7 days in an EDM-1 differentiation system, carrying out genotype identification, and determining mutation efficiency; EDM-2 stage differentiation was performed for 4 days after mutation was confirmed, and EMD-3 stage differentiation was performed for 7 days. And extracting RNA after differentiation of each stage, reverse transcribing into cDNA gel electrophoresis to detect abnormal splicing of the intron, and verifying whether the CDS region of the restored hematopoietic stem cells can restore normal coding functions.
In another aspect, the invention provides a method for repairing an abnormal splicing of an intron caused by an IVS 2-254C > T mutation of HBB (beta-globin gene), wherein the sgRNA comprises a targeting sequence CAGTGATAATTTCTGGGTTA of the sgRNA-1.
Further, the sgRNA also comprises the sgRNA-2, and the targeting sequence of the sgRNA-2 is TAAATTGTAACTGATGTAAG.
On the other hand, the invention also provides application of the sgRNA in repairing abnormal splicing of introns caused by HBB (beta-globin gene) IVS 2-650C > T mutation in cells.
Further, the cells are hematopoietic stem cells, preferably, the cells are CD34 + Is selected from the group consisting of hematopoietic stem and progenitor cells; more preferably, the cell is an ex vivo cell.
The beneficial effects are that:
the invention designs sgRNA for non-targeted regions of IVS2-654C > T, which provides possibility for more accurate and flexible editing on genome, and the mutation efficiency of the invention can reach up to 95% which is significantly higher than mutation rate achieved by ZFN, TALEN or Cas12a/Cpf1 RNP. The invention has great significance in saving experiment time and investment of manpower and material resources through the realized high mutation efficiency.
According to the invention, a method for quickly constructing mutation near the pathogenic site of the hematopoietic stem cells of a beta-Dilean IVS2-654C > T patient is adopted, guide RNA capable of cutting the pathogenic site and CAS9 protein are directly introduced into the defective hematopoietic stem cells, and the frame shift of a target gene is caused by DNA double strand break (double strand break, DSB) and Non-homologous end joining (Non-homologous end joining, NHEJ) repair caused by the DNA double strand break is carried out, so that the pathogenic site can be quickly and efficiently destroyed. The beta-globin intron obtained by the invention can be spliced normally, and the CDS region restores the coding function.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:
fig. 1 is a schematic diagram of the principle of action of a CRISPR/Cas9 system.
FIG. 2 is a schematic representation of the beta-barren IVS 2-650C > T aberrant splice mutation site.
FIG. 3 is a schematic representation of Sanger sequencing by extracting genomic DNA from cells and PCR amplifying the corresponding genomic region after editing the pathogenic sites of hematopoietic stem cells of a patient, wherein the upper panel is an electrotransfer blank control group (IVS 2-654 patient-derived hematopoietic stem cells, without any RNP introduced), and the sequencing peaks are normal; the middle panel is the electrotransport sgRNA-1 panel, and the sequencing peak plot shows the generation of hetero-peaks from the cutting site of the sgRNA-1 due to the loss of random numbers of bases; the lower panel is the electrotransformed sgRNA-1+sgRNA-2 set and the sequencing peak plot shows the generation of hetero-peaks from the cutting site of the sgRNA-1 due to the loss of random numbers of bases.
FIG. 4 shows the PCR detection gel electrophoresis of cDNA after editing the pathogenic sites of hematopoietic stem cells and differentiating erythrocytes of a patient. The upper band (468 bp) with slower migration is an abnormal sheared amplification product, the lower band (395 bp) with slower migration is a normal sheared amplification product, and the ratio of the abnormal sheared to normal sheared band intensity of the edited sample is obviously reduced. Lanes 1 and 6 are DNA molecular size standards, lanes 2-5 are samples of red blood cells differentiated in vitro to 11 days (in EDM-2 medium), and lanes 7-10 are samples of red blood cells differentiated in vitro to 18 days (in EDM-3 medium). HU-2 represents in vitro differentiated erythrocytes of healthy human origin, IVS2-654 represents in vitro differentiated erythrocytes of poor patient origin, 654-sg1 or 654-sg1+2 represents in vitro differentiated erythrocytes after Cas9 editing.
FIG. 5 shows PCR amplification of HBB sequences from intact cDNA after editing of hematopoietic stem cell pathogenic sites and erythrocyte differentiation in a patient and Sanger sequencing; the upper panel is an electrotransfer blank (IVS 2-654 patient-derived hematopoietic stem cells without any RNP introduced), and the sequencing peak shows that a double peak is generated at the splice of the second and third exons due to one allele splice abnormality; the middle graph is an electrotransfer sgRNA-1 group, and the sequencing peak graph shows that the main peak is a normal HBB sequence, and only has double peaks caused by extremely low shearing abnormality; the lower panel shows the electrotransferred sgRNA-1+sgRNA-2 group, and the sequencing peak shows the main peak as normal HBB sequence, and only has the double peak caused by extremely low shear abnormality.
FIG. 6 is a schematic representation of globin qPCR after mutation differentiation of hematopoietic stem cell pathogenic sites in a patient. The results showed that the ratio of mRNA for HBB and HBA was approximately 50% for cells edited with sgRNA-1, whereas the ratio of mRNA for HBB and HBA was approximately 100% for cells edited with sgRNA-1+2. The ratio is sufficient to eliminate erythrotoxicity caused by excessive HBA level, and can effectively relieve thalassemia symptoms.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, to which the present invention is not limited. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims. The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited. Such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or in accordance with the manufacturer's recommendations.
The invention utilizes CRISPR-Cas9 gene editing technology shown in figure 1 to target and destroy abnormal mutation sites IVS2-654C > T (figure 2) in beta-thalassemia, and constructs a guide RNA sequence (sgRNA) capable of recognizing and guiding Cas9 protein to target gene target sequence, which is a method for targeting and changing pathogenic target DNA, and comprises the following steps: and introducing sgRNA encoding nucleic acid for identifying target genes and Cas9 protein into the defective hematopoietic stem cells, so as to identify and cut target genomic DNA sequences. The cells are then cultured in vitro to express the nuclease and double strand break the target genomic DNA near the pathogenic site, followed by repair of the DNA break site.
The repairing method comprises the following steps: (a) non-homologous end joining repair. Non-homologous end joining repair results in the introduction of a genetic mutation (base insertion, deletion) into the genomic sequence of interest. (b) homologous recombination repair. Homologous recombination repair causes the introduction of a donor exogenous DNA sequence into the target genomic DNA sequence, resulting in a change in the endogenous target gene sequence. In this embodiment, no exogenous DNA sequence need be introduced.
The invention relates to a construction method for destroying pathogenic sites in IVS2-654C > T defective hematopoietic stem cells, which comprises the following steps:
(1) sgRNA design:
sgRNA-1 targeting sequence CAGTGATAATTTCTGGGTTA (SEQ ID No. 1);
sgRNA-2 targeting sequence TAAATTGTAACTGATGTAAG (SEQ ID No. 2);
(2) sgRNA synthesis;
(3) The sgRNA and the Cas9 protein are mixed according to a molar ratio of 1-2:1 mixing and electrotransducing into hematopoietic stem cells of a β -thalassemia IVS 2-650 c > t patient;
(4) Culturing the cells subjected to electrotransformation in a CD34+EDM-1 differentiation system for 7 days, extracting genome DNA of the cells obtained in the steps, carrying out genotype identification, and determining mutation efficiency;
(5) EDM-2 stage differentiation was performed for 4 days after mutation was confirmed, and EMD-3 stage differentiation was performed for 7 days. And extracting RNA after differentiation at each stage, detecting abnormal splicing of introns by reverse transcription into cDNA gel electrophoresis, detecting the content and the proportion of edited globin mRNA by qPCR, and verifying whether the CDS region of the hematopoietic stem cells can restore normal coding functions.
In the present invention, hematopoietic stem cells refer to beta-barren IVS2-654C>Hematopoietic stem/progenitor cells of T-genotype patient (C)D34 + HSPCs). Electrotransformation of the chemically modified sgrnas and Cas9 protein mixture to β -barren IVS2-654C>T hematopoietic stem cells, which efficiently disrupt aberrant splice mutation sites, thereby restoring expression of the beta-globin gene. The invention can repair the beta-barren IVS2-654C dependent on the blood transfusion type by using the existing gene editing technology>T, the editing efficiency is high, and the lasting balance hematopoietic system of autologous hematopoietic stem cells can be modified with high efficiency.
EXAMPLE 1 disruption of pathogenic sites in beta-Dilean IVS 2-650C > T-deficient hematopoietic Stem cells
1. Design of sgRNA
In this example, the patient is a beta-thalassemia double heterozygote, the genotype is IVS2-654/CD17, and the example is based on the fact that the pathogenic site of IVS2-654 does not have a suitable PAM targeting cleavage to destroy abnormal splice sites, so that sgRNA-1 and sgRNA-2 are designed before and after the pathogenic site respectively. Wherein, the single action of the sgRNA-1 can cause fracture repair before the pathogenic mutation site, and the combined action of the sgRNA-1 and the sgRNA-2 can generate large fragment deletion before and after the pathogenic site.
2. Preparation of sgRNA and Cas9 proteins
3. Electrolysis of sgrnas and Cas9 protein complexes
Mixing chemically modified synthesized sgRNA and Cas9 protein according to a certain ratio, incubating at room temperature for 10min, respectively electrotransferring sgRNA-1 (654-sg 1), sgRNA-1+sgRNA-2 (654-sg1+2), blank control (cells without adding any RNP), mixing electrotransferring solutions according to the ratio of electrotransferring kit, and electrotransferring cell number not exceeding 10 5 And re-suspending the cells by using electrotransfer liquid after centrifuging the cells, slightly mixing the cells with the incubated sgRNA and Cas9 protein complex, transferring the mixture to an electrotransfer cup, avoiding generating bubbles in the operation process, performing electrotransfer (Lonza-4D electrotransfer instrument) by using CD34 cell electrotransfer program EO-100, standing at room temperature for incubation for 5min after confirming that the electrotransfer is successful, and re-centrifuging to remove Cas9 protein and electrotransfer liquid and CD34 + After the EDM-1 culture medium is used for resuspension of cells, the cells are added into a cell culture plate for 37-degree differentiation culture, and the damage of the invention on pathogenic mutation sites of the defective hematopoietic stem cells is completed.
The following was used to identify whether the method of constructing the target gene mutation of the present invention was successfully carried out.
(1) Mutation identification of genomic DNA
And (3) after the hematopoietic stem cells obtained in the step (3) are subjected to in vitro differentiation culture for 3-4 days, collecting a proper amount of cells, extracting genome, detecting mutation efficiency by Sanger sequencing after PCR amplification, and continuously differentiating the rest cells in an EDM-1 culture medium until the 7 th day. As shown in FIG. 3, the mutation rate of the target site after electrotransformation can be as high as 95%. Wherein, 654-check-F primer sequence: CACATATTGACCAAATCAGGG (SEQ ID No. 3); 654-check-R primer sequence: CTTTGCCAAAGTGATGGGCCA (SEQ ID No. 4).
(2) EDM-2 and EDM-3 differentiation culture
After Sanger sequencing, the mutation of the target site is determined to be successful, the mutation can be continued in the EDM-2 culture medium. The cells can be greatly amplified at this stage, when the differentiation stage of EDM-2 is finished, a proper amount of cells are collected, RNA is extracted and reversely transcribed into cDNA, meanwhile, the rest cells continue to differentiate until EDM-3, and RNA is extracted and reversely transcribed into cDNA after the differentiation is finished.
(3) Exon, CDS region amplification, q-PCR
And amplifying the differentiated exons, and verifying whether the mutation of the pathogenic site can be spliced normally. As shown in fig. 4, the hematopoietic stem cell introns after the pathogenic site mutation restored substantially normal splicing compared to the defective cells; as shown in FIG. 5, the sequencing result shows that compared with the defective cells, the hematopoietic stem cell HBB gene with the mutation of the pathogenic site restores the normal coding function. q-PCR analysis of changes in the globin content of hematopoietic stem cells following site mutation As shown in FIG. 6, the mRNA ratio of HBB and HBA was approximately 50% in cells edited with sgRNA-1, whereas the mRNA ratio of HBB and HBA was approximately 100% in cells edited with sgRNA-1+2. The ratio is sufficient to eliminate erythrotoxicity caused by excessive HBA level, and can effectively relieve thalassemia symptoms.
Wherein, the primer sequences used are as follows:
654-exon1-F primer sequence: TGAGGAGAAGTCTGCCGTTAC (SEQ ID No. 5);
654-exon3-R primer sequence: CACCAGCCACCACTTTCTGA (SEQ ID No. 6);
HBB-CDS-F primer sequence: ATGGTGCATCTGACTCCTGA (SEQ ID No. 7);
HBB-CDS-R primer sequence: TTAGTGATACTTGTGGGCCA (SEQ ID No. 8);
HBA-S_qPCR primer sequence: GCCCTGGAGAGGATGTTC (SEQ ID No. 9);
HBA-A_qPCR primer sequence: TTCTTGCCGTGGCCCTTA (SEQ ID No. 10);
HBB-S_qPCR primer sequence: TGAGGAGAAGTCTGCCGTTAC (SEQ ID No. 11);
HBB-AS_qPCR primer sequence: ACCACCAGCAGCCTGCCCA (SEQ ID No. 12);
HBG-S_qPCR primer sequence: GGTTATCAATAAGCTCCTAGTCC (SEQ ID No. 13);
HBG-AS_qPCR primer sequence: ACAACCAGGAGCCTTCCCA (SEQ ID No. 14);
hbb_e2-e3 primer sequence: TTCAGGCTCCTGGGCAAC (SEQ ID No. 15);
r_hbb_exon3 primer sequence: CACCAGCCACCACTTTCTGA (SEQ ID No. 16).
The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the claims of the present application.
SEQUENCE LISTING
<110> Shanghai Yao Biotech Co., ltd, university of Huadong
<120> a method for repairing abnormal splicing of introns
<130> JH-CNP190598
<160> 16
<170> PatentIn version 3.5
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Claims (6)
1. Use of a CRISPR-Cas9 system for repairing abnormal splicing of introns in cells due to mutations in the β -globin gene IVS2-654c > t, said mutations in IVS2-654c > t introducing additional splice donor sites leading to abnormal splicing of introns, for the preparation of a cell therapeutic preparation, characterized in that:
the CRISPR-Cas9 system carries out gene editing on beta-globin genes;
the CRISPR-Cas9 system comprises Cas9 and sgrnas targeting a target sequence;
the sgRNA of the target sequence is as follows:
(1) sgRNA-1 with a targeting sequence of CAGTGATAATTTCTGGGTTA; or (b)
(2) A targeting sequence of CAGTGATAATTTCTGGGTTA for sgRNA-1, and TAAATTGTAACTGATGTAAG for sgRNA-2;
the targeting site of the sgRNA is in the range of 20bp upstream of the IVS2-654C > T site to 70bp downstream of the IVS2-654C > T site;
the cell therapy is cell therapy aiming at thalassemia, and the cells are hematopoietic stem cells.
2. The use according to claim 1, wherein the sgRNA comprises chemical modification of bases.
3. The use according to claim 1 or 2, characterized in that the complex comprising Cas9 and sgRNA is introduced into the cell by means of electrotransformation.
4. The use according to claim 1 or 2, wherein the cell is CD34 + Is a hematopoietic stem progenitor cell of (a).
5. A set of sgrnas for repairing aberrant splicing of introns caused by an IVS2-654c > t mutation of a β -globin gene, said set of sgrnas consisting of:
the targeting sequence of the sgRNA-1 is shown as SEQ ID No. 1;
and the targeting sequence of the sgRNA-2 is shown as SEQ ID No. 2.
6. The use of the sgRNA panel of claim 5 in the preparation of a medicament, said medicament being: a medicament for treating a disease caused by mutation of the beta-globin gene IVS2-654C > T, wherein the disease is thalassemia.
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