CN112391410A

CN112391410A - sgRNA and application thereof in repairing abnormal splicing of intron

Info

Publication number: CN112391410A
Application number: CN202010097964.8A
Authority: CN
Inventors: 吴宇轩; 杨菲; 刘明耀; 张亮
Original assignee: Shanghai Bangyao Biological Technology Co ltd; East China Normal University
Current assignee: Shanghai Bangyao Biological Technology Co ltd; East China Normal University
Priority date: 2020-02-17
Filing date: 2020-02-17
Publication date: 2021-02-23
Anticipated expiration: 2040-02-17
Also published as: CN112391410B

Abstract

The application discloses a sgRNA and application thereof in repairing abnormal splicing of introns. The sgRNA is used for repairing intron abnormal splicing caused by HBB (beta-globin gene) IVS2-654C > T mutation, and comprises sgRNA-U and sgRNA-D, wherein the targeting site of the sgRNA-U is in the range from 21bp upstream of IVS2-654C > T site to the second intron starting site of the HBB gene in which IVS2-654C > T site is positioned, and the targeting site of the sgRNA-D is in the range from 71bp downstream of IVS2-654C > T site to the second intron termination site of the HBB gene in which IVS2-654C > T site is positioned. The invention can repair the blood transfusion dependent beta-thalassemia IVS2-654C > T by using the existing gene editing technology, has high editing efficiency and can efficiently modify the autologous hematopoietic stem cell persistent balance hematopoietic system.

Description

sgRNA and application thereof in repairing abnormal splicing of intron

Technical Field

The invention relates to the technical field of genetic engineering, in particular to sgRNA and application thereof in repairing abnormal splicing of introns.

Background

Beta-thalassemia is a common hereditary disease with abnormal hemoglobin in adults caused by beta-globin gene defects, and about 3000 thousands of people are gene carriers of thalassemia in China, and about 3000 thousands of families and 1 hundred million people are involved, wherein about 30 thousands of patients with severe thalassemia and intermediate thalassemia are involved. The IVS2-654C > T genotype is a common one in 'poor land' in China, and the pathogenesis is that the 654 th base in the 2 nd intron of the HBB gene generates C > T mutation, so that an abnormal splice site is generated, 73nt of exon is additionally added in beta-globin mRNA, and translation is terminated in advance.

At present, patients with intermediate and severe thalassemia need long-term blood transfusion and deferrization treatment to maintain life, the only radical treatment mode is allogeneic hematopoietic stem cell transplantation, but the main implementation obstacles are the shortage of blood resources in China, the allogenic hematopoietic stem cell matching difficulty, transplantation related complications and the like. Among them, gene therapy using lentiviral vectors has shown great potential, but the semi-random vector integration is at risk of carcinogenesis. Meanwhile, expression elements in lentiviruses are gradually silenced in the long-term homing and self-renewal process of hematopoietic stem cells, so that the curative effect is reduced, and the aim of lifelong healing cannot be fulfilled. In addition, the high concentration and quality of lentivirus required clinically is extremely demanding in terms of equipment and technology, and therefore cost reduction is difficult. Therefore, a parallel, safer, less costly clinical protocol is highly desirable.

The ideal method of gene therapy is to repair or destroy the classical anergy mutations in the diseased human hematopoietic stem cell DNA, to restore gene function and to produce permanently wild-type adult β -globin under the action of endogenous transcriptional control factors, to differentiate normally into erythroid cells. The repair mode of the DNA sequence after gene editing mainly comprises Non-homologous end joining (NHEJ) repair, the proportion of homologous recombination mediated repair (HDR) is extremely low, and efficient HDR efficiency is required for repairing point mutation.

Aiming at the abnormal mutation of the pathogenic site, the strategy that the mutation site can be destroyed only by cutting the target DNA in a targeted manner to realize NHEJ mutation is more feasible. Clinically, the aim of healing can be achieved only by transplanting autologous hematopoietic stem cells repaired in an NHEJ mode after gene editing back into the body.

Disclosure of Invention

The invention provides a sgRNA and a repair method thereof for abnormal splicing of an intron caused by mutation of a beta-globin gene (HBB) IVS2-654C > T.

The invention repairs the abnormal splicing of the intron caused by IVS2-654C > T by using a CRISPR-Cas9 system. When Cas9 targets the cleavage site, it is necessary that the 3 bases immediately adjacent to the 3 ' end of the target must be in the form of 5 ' -NGG-3 ' in the selection of sequences for targeting, in order to constitute a 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 IVS2-654C > T mutation site using the CRISPR-Cas9 system.

Intensive studies by the inventors on the aberrant splicing of introns by IVS2-654C > T revealed that mutation of IVS2-654C > T resulted 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 bases in excess, leading to premature termination of translation. In theory, disruption or deletion of the extra splice donor site resulting from IVS2-654C > T in the HBB gene region, such that it does not initiate aberrant variable splicing, allows this genomic region to be transcribed to produce normal β -globin mRNA and translated to produce normal β -globin.

In one embodiment, the pathogenic site IVS2-654C > T is used as a boundary, the CRISPR-Cas9 system can be used to simultaneously target and cut the upstream and downstream ends of the whole intron or the vicinity of the upstream and downstream ends of the whole intron respectively, and a large fragment of the gene region containing the pathogenic site is deleted along with the insertion, deletion, change, frame shift mutation or knockout of the base, namely, the additional splice donor site "AAGGTAATA" caused by IVS2-654C > T can be directly and efficiently destroyed or deleted.

In the invention, the CRISPR-Cas system refers to a CRISPR-Cas system suitable for being artificially modified and a nuclease system derived from an archaebacterium II type (CRISPR) -CRISPR-associated protein (Cas) system, and compared with ZFN and TALEN, the system is simpler and more convenient to operate.

The invention adopts RNA-guided endonucleases (RGENs) to realize specific cutting of a target gene sequence. RGENs are composed of chimeric guide RNA and Cas9 protein, wherein the former is formed by fusing CRISPR RNAs (crRNAs) and trans-activating crRNAs (tracrRNAs) in a naturally-occurring II-type CRISPR-Cas system into single-stranded guide RNA (sgRNA), so as to combine with Cas9 protein and guide the latter to perform specific cleavage on two target DNA sequences upstream and downstream of a pathogenic site simultaneously, and the cleavage will form double-strand break (DSB), and the damage can cause the deletion of a large DNA fragment including the pathogenic site, thereby realizing the damage or deletion of the pathogenic mutation site.

The Cas9 may be selected from Streptococcus pyogenes, Staphylococcus aureus or n. The Cas9 may be selected from a wild-type Cas9, and may also be selected from a mutant-type Cas 9; the mutant Cas9 did not result in loss of cleavage and targeting activity of Cas 9.

In other embodiments, other Cas enzymes may be used in place of Cas 9.

In the invention, the pathogenic site IVS2-654C > T is used as a boundary, a CRISPR-Cas9 system is adopted to respectively target the upstream end and the downstream end, namely the whole intron range for cutting, and a large fragment of a gene region of the intron region containing the pathogenic site IVS2-654C > T is deleted along with the modes of base insertion, deletion, change, frame shift mutation or knockout, so that the additional spliced donor site AAGGTAATA caused by the IVS2-654C > T can be directly and efficiently destroyed or deleted, thereby achieving the repairing effect. Therefore, in the present invention, Cas9 does not need to introduce a foreign DNA donor sequence for homologous recombination repair after cleavage of the target sequence.

In one embodiment, the cleavage of the target sequence is performed using a combination of sgRNA-U and sgRNA-D, the sgRNA-U having a targeting sequence of 5'-TTCTTTCCCCTTCTTTTCTA-3' (SEQ ID No.1) and the sgRNA-D having a targeting sequence of 5'-GATTATTCTGAGTCCAAGCT-3' (SEQ ID No.36), the sgRNA-U and the sgRNA-D can direct Cas9 to simultaneously cleave DNA upstream and downstream of the pathogenic site IVS2-654C > T, respectively, resulting in the loss of large fragment bases including the pathogenic site, thereby resulting in the efficient disruption and deletion of the additional splice donor site due to IVS2-654C > T, resulting in the loss of function of the additional splice donor site.

Since such poor mutation sites of IVS2-654C > T occur in the intronic gene region, it is theorized that the function of the additional splice donor site can be disrupted so that it does not initiate aberrant variable splicing, so that the genomic region can be transcribed to produce normal β -globin mRNA and translated to produce normal β -globin, if only the partially pathogenic splice donor is disrupted or deleted.

The experimental result shows that in the second intron region of the normal human HBB, if the upstream and downstream end ranges are targeted respectively by taking the pathogenic site as a boundary for gene editing of sgRNA + D, the extra splice donor large fragment DNA containing the pathogenic site is cut and deleted, and the normal splicing or transcription of the beta-globin mRNA cannot be influenced. This indicates that when editing a disease-causing allele, 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 repair method for aberrant splicing of introns caused by mutation of HBB (β -globin gene) IVS2-654C > T in a cell, which IVS2-654C > T may cause an additional splice donor site, the method comprising the step of gene editing HBB using the CRISPR-Cas9 system to delete the additional splice donor site;

the CRISPR-Cas9 system includes Cas9 and sgrnas that target a target sequence;

the sgRNA includes sgRNA-U and sgRNA-D,

the targeting site of the sgRNA-U is within the range from 21bp upstream of the IVS2-654C > T site to the initiation site of the second intron of the HBB gene in which the IVS2-654C > T site is positioned,

the targeting site of the sgRNA-D is within a range from 71bp downstream of the IVS2-654C > T site to the termination site of the second intron of the HBB gene in which the IVS2-654C > T site is located.

Further, the targeting sequence of the sgRNA-U is any one or more than two of the sequences shown in SEQ ID No. 1-35; preferably, the sequence shown in SEQ ID nos. 1, 2, and/or 3, and more preferably, the sgRNA-U includes a chemical modification of a base.

Further, the targeting sequence of the sgRNA-D is any one or more than two of the sequences shown in SEQ ID No. 36-51; preferably, the sequence shown as SEQ ID No.36, 37, 38 and/or 39; more preferably, the sgRNA-D comprises a chemical modification of a base.

Further, the sequence of the additional splice donor site is AAGGTAATA.

Further, a complex comprising Cas9 and sgRNA-U, and a complex of Cas9 and sgRNA-D were introduced into the cells using an electrotransformation method.

Further, the cells are hematopoietic stem cells or erythroid progenitor cells,

preferably, the hematopoietic stem cell is CD34⁺The hematopoietic stem progenitor cells of (1), said erythroid progenitor cells being HUDEP-2,

more preferably, the cell is an ex vivo cell.

In one embodiment, the sgRNA includes a chemical modification of a base. In a preferred embodiment, the sgRNA includes a chemical modification of any one or any several of the 1 st to n th bases at the 5 'end, and/or a chemical modification of any one or any several of the 1 st to n th bases at the 3' end; and n is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, the sgRNA comprises a chemical modification of one, two, three, four or five bases at the 5 'end and/or a chemical modification of one, two, three, four or five bases at the 3' end. For example, the sgRNA is chemically modified at the 1 st, 2 nd, 3 rd, 4 th, 5 th, or 1 to 2 nd, 1 to 3 rd, 1 to 4 th, and 1 to 5 th bases at the 5' end of the sgRNA; and/or chemically modifying the 1 st base, the 2 nd base, the 3 rd base, the 4 th base, the 5 th base, 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 at 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, when introducing into the cell a complex comprising Cas9 and sgRNA-U and a complex of Cas9 and sgRNA-D by means of electrotransformation, the molar ratio of Cas9 and sgRNA-U (or sgRNA-D) is 1:1-3, preferably 1:2, more preferably 1: 3.

Further, the Cas9 and sgRNA-U (or sgRNA-D) 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 ratio of the complex comprising Cas9 and sgRNA-U (or sgRNA-D) to the amount of cells is 20-100 μ g of complex: (1X 10)²-1×10⁶Individual) cells, preferably, 30 μ g of complex: (1X 10)³-1×10⁵One) cells.

Further, the cells after electroporation were cultured in CD34⁺Extracting the genome DNA of the cell obtained in the step for 7 days in an EDM-1 differentiation system, and performing gel electrophoresis after PCR for identification; after deletion of large fragment base is determined, EDM-2 stage differentiation is carried out for 4 days, and EMD-3 stage differentiation is carried out for 7 days. After the differentiation is finished, RNA is extracted, reverse transcription is carried out to detect abnormal splicing of the intron by cDNA gel electrophoresis, and whether the CDS region of the restored hematopoietic stem cell can recover the normal coding function is verified.

In another aspect, the invention also provides a sgRNA for repairing an intron splice abnormality caused by an HBB (beta-globin gene) IVS2-654C > T mutation, the sgRNA including sgRNA-U and sgRNA-D,

the targeting site of the sgRNA-U is within the range from 21bp upstream of the IVS2-654C > T site to the initiation site of the second intron of the HBB gene in which the IVS2-654C > T site is located;

Further, the targeting sequence of the sgRNA-U is any one or more than two of the sequences shown in SEQ ID No. 1-35; preferably, the sequence shown in SEQ ID nos. 1, 2, and/or 3, more preferably, the sgRNA-U comprises a chemical modification of a base;

the targeting sequence of the sgRNA-D is any one or more than two of the sequences shown in SEQ ID No. 36-51; preferably, the sequence shown as SEQ ID No.36, 37, 38 and/or 39; more preferably, the sgRNA-D comprises a chemical modification of a base.

On the other hand, the invention also provides application of the sgRNA in repairing intron abnormal splicing caused by HBB (beta-globin gene) IVS2-654C > T mutation in cells.

The cells are hematopoietic stem cells or erythroid progenitor cells,

preferably, the hematopoietic stem cell is CD34⁺The hematopoietic stem progenitor cells of (a), said erythroid progenitor cells are HUDEP-2;

more preferably, the cell is an ex vivo cell.

Has the advantages that:

the sgRNA is designed aiming at the second intron region of the HBB gene where IVS2-654C > T is located, so that the possibility of more accurate and flexible editing on a genome is provided, the mutation efficiency of the invention can reach 95%, and the deletion of large-fragment DNA in the intron gene region is obviously higher than the mutation rate which can be achieved by adopting ZFN, TALEN or Cas12a/Cpf1 RNP. The method has great significance in saving experiment time and investment of manpower and material resources through the realized high mutation efficiency.

The invention relates to a method for rapidly constructing mutation from upstream and downstream of a pathogenic site of a hematopoietic stem cell of a beta-thalassemia IVS2-654C > T patient to two ends of a second intron, directly introducing guide RNA and CAS9 protein which can delete the pathogenic site into a defective hematopoietic stem cell, and rapidly and efficiently causing the damage and deletion of the pathogenic site through Double Strand Break (DSB). The beta-globin intron obtained by the invention can be normally spliced, 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 embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

fig. 1 is a schematic diagram of the CRISPR/Cas9 system action principle.

FIG. 2 is a schematic representation of the aberrant splicing mutation sites of β -thalassemia IVS2-654C > T.

FIG. 3 is a gel electrophoresis diagram of PCR amplification of a target sequence in genomic DNA of hematopoietic stem cells of a patient. Wherein, from left to right, lane 1 is the DNA size standard, and lane 2 is the amplified fragment of blank control (1388 bp); lane 3 is an amplified fragment of sg-U + D.

FIG. 4 is a gel electrophoresis image of HBB exon in patient hematopoietic stem cell cDNA amplified by PCR. From left to right, lane 1 shows the size standard of DNA molecules, and lanes 2-4 show the samples of erythrocytes differentiated in vitro for 18 days. 654 represents a poor patient-derived in vitro differentiated erythrocyte, sg-U + D represents an in vitro differentiated erythrocyte after Cas9 editing, CD34⁺In vitro differentiated erythrocytes representing a healthy human origin, the upper slower migrating band A (468bp) is the aberrantly spliced amplification product and the lower faster migrating band N (395 bp) is the normally spliced amplification product.

FIG. 5 shows the sequencing result of the CDS region of HBB of hematopoietic stem cells of patients amplified by PCR. Wherein the top panel is a blank control result and the sequencing peak panel shows that a doublet is generated at the splice of the second and third exons due to the abnormal splice of one allele; the middle graph is the result of electrotransformation of sg-U + D, and the sequencing peak graph shows that the main peak is a normal HBB sequence and is basically free of double peaks caused by abnormal shearing; the lower panel shows hematopoietic stem cells derived from healthy humans.

FIG. 6 is a diagram of globin qPCR after differentiation of hematopoietic stem cell pathogenic site mutation of a patient. Wherein 654 represents the result of ex vivo differentiated erythrocytes derived from poor patients, and sg-U + D represents the result of in vitro differentiated erythrocytes edited by Cas 9.

FIG. 7 is a gel electrophoresis of PCR amplification of a target sequence in genomic DNA from HUDEP-2 cells. Wherein, from left to right, lane 1 is a blank control result; lanes 2-5 are the results of treatments 1-4, respectively.

FIG. 8 is a gel electrophoresis of HBB exons in PCR amplified HUDEP-2 cell cDNA. Wherein, from left to right, lane 1 is the DNA size standard, and lane 2 HUDEP-2 represents the blank control result; lanes 3-6 deal with the results of lanes 1-4, respectively.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents 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 according to the manufacturer's recommendations.

The invention relates to a method for targeting and changing pathogenic target DNA (deoxyribonucleic acid), which aims to destroy abnormal mutation sites IVS2-654C > T in beta-thalassemia by using a CRISPR-Cas9 gene editing technology (shown in figure 2) and construct a guide RNA sequence (sgRNA) capable of identifying and guiding Cas9 protein to a target gene target sequence, and the method comprises the following steps: two sgRNA-encoding nucleic acids for identifying target genes and a Cas9 protein are introduced into defective hematopoietic stem cells, and the target genomic DNA sequence is simultaneously identified and cleaved (as shown in fig. 1). Then, the cells are cultured in vitro, so that nuclease is expressed and double strand breaks occur in the target genomic DNA at both ends of the pathogenic site (in the second intron gene region), resulting in deletion of a large fragment including the pathogenic site. In this embodiment, no foreign DNA sequences need to 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) designing an sgRNA;

(2) synthesizing sgRNA;

(3) the sgRNA and Cas9 proteins were mixed in a molar ratio of 1-2: 1 mixing and electrotransfering hematopoietic stem cells of a patient with beta-thalassemia IVS2-654C > T;

(4) the cells after electroporation were cultured in CD34⁺Extracting the genome DNA of the cell obtained in the step for 7 days in an EDM-1 differentiation system, and performing PCR amplification to determine the mutation efficiency;

(5) after the mutation is determined, EDM-2 stage differentiation is carried out for 4 days, and EMD-3 stage differentiation is carried out for 7 days. After the differentiation is finished, extracting RNA, carrying out reverse transcription to obtain cDNA, detecting abnormal splicing of an intron by gel electrophoresis and detecting the content and proportion of the mRNA of the globin after editing by qPCR, and verifying whether the CDS region of the restored hematopoietic stem cell can recover the normal coding function.

In the present invention, the hematopoietic stem cells are beta-thalassemia IVS2-654C>T genotype patient hematopoietic stem progenitor cells (CD 34)⁺HSPCs). The chemically modified sgRNA and Cas9 protein mixture is electrically transformed to beta-thalassemia IVS2-654C>T hematopoietic stem cells, and the abnormal splicing mutation sites are efficiently destroyed and deleted, thereby restoring the expression of the beta-globin gene. The invention can repair the transfusion dependent type beta-thalassemia IVS2-654C by using the existing gene editing technology>T, the editing efficiency is high, and the autologous hematopoietic stem cells can be efficiently modified to permanently balance the hematopoietic system.

Example 1 design of sgRNA

Designing sgRNA, named sgRNA-U, with the code numbers of sgRNA-U1, 2, 3 and … …, based on a sequence from 21bp upstream of the IVS2-654C > T pathogenic site to the initiation site range of a second intron of an HBB gene where the IVS2-654C > T site is located;

based on a sequence from downstream 71bp of IVS2-654C > T pathogenic site to termination site of second intron of HBB gene where IVS2-654C > T site is located, sgRNA is designed and named as sgRNA-D, and the sequence numbers are sgRNA-D1, 2, 3 and … ….

The interaction of one sgRNA-U and one sgRNA-D leads to the deletion of the corresponding fragment in front of and behind the pathogenic site.

The targeting sequences of the sgRNA-U and sgRNA-D are shown in Table 1.

TABLE 1

Example 2 disruption of pathogenic sites in beta-thalassemia IVS2-654C > T deficient hematopoietic stem cells

The beta-thalassemia IVS2-654C > T defective hematopoietic stem cells used in this example were from a double heterozygous patient with a genotype of IVS2-654C > T complex CD17 mutation.

First, destroy method

1. Preparation of sgRNA

Chemical modification synthesis a total of 51 sgRNA-U and sgRNA-D in table 1.

2. Electrotransformation of sgRNA and Cas9 protein complexes

Mixing sgRNA-U or sgRNA-D synthesized by chemical modification and Cas9 protein according to a certain proportion, incubating for 10min at room temperature, and simultaneously electrically converting different combinations of sgRNA-U and sgRNA-D (hereinafter referred to as sg-U + D) to beta-poor IVS2-654C>T-deficient hematopoietic stem cells and beta-diji IVS2-654C without any RNP addition>T-deficient hematopoietic stem cells ofBlank control, mixing electrotransfer liquid according to the proportion of electrotransfer kit, the number of electrotransfer cells is not more than 10⁵And then, after the cells are centrifuged, the cell is resuspended in an electrotransformation solution and is gently mixed with the incubated sgRNA and Cas9 protein compound, the mixture is transferred to an electrotransfer cup, bubbles are avoided in the operation process, a CD34 cell electrotransfer program EO-100 is used for electrotransfer (Lonza-4D electrotransfer), after the successful electrotransfer is confirmed, the cells are stood and incubated for 5min at room temperature, the Cas9 protein and the electrotransfer solution are removed by recentrifugation, and the CD34⁺EDM-1 culture medium suspending cells, adding into cell culture plate at 37 deg.C and 5% CO₂And performing differentiation culture in an incubator to complete the destruction and deletion of pathogenic mutation sites of the defective hematopoietic stem cells.

The combination of the electrotransformed sgRNA-U and sgRNA-D is as follows:

sgRNA-U1 (or sgRNA-U16 or sgRNA-U32) and sgRNA-D1,

sgRNA-U2 (or sgRNA-U17 or sgRNA-U33) and sgRNA-D2,

sgRNA-U2 (or sgRNA-U18 or sgRNA-U34) and sgRNA-D3,

sgRNA-U3 (or sgRNA-U19 or sgRNA-U35) and sgRNA-D4,

sgRNA-U4 (or sgRNA-U20) and sgRNA-D5,

sgRNA-U5 (or sgRNA-U21) and sgRNA-D6,

sgRNA-U6 (or sgRNA-U22) and sgRNA-D7,

sgRNA-U7 (or sgRNA-U23) and sgRNA-D8,

sgRNA-U8 (or sgRNA-U24) and sgRNA-D9,

sgRNA-U9 (or sgRNA-U25) and sgRNA-D10,

sgRNA-U10 (or sgRNA-U26) and sgRNA-D11,

sgRNA-U11 (or sgRNA-U27) and sgRNA-D12,

sgRNA-U12 (or sgRNA-U28) and sgRNA-D13,

sgRNA-U13 (or sgRNA-U29) and sgRNA-D14,

sgRNA-U14 (or sgRNA-U30) and sgRNA-D15,

sgRNA-U15 (or sgRNA-U31) and sgRNA-D16.

II, identification result

1. Mutation identification of genomic DNA

The method comprises the following steps: and (3) carrying out in-vitro differentiation culture on the hematopoietic stem cells obtained in the step (2) in the step (I) for 3-4 days, collecting a proper amount of cells, extracting a genome, carrying out gel electrophoresis detection on an amplification product after PCR amplification, identifying the mutation efficiency and the deletion condition of the DNA fragment, and continuously differentiating the rest cells in an EDM-1 culture medium until the 7 th day.

Wherein, the primer sequences used in the PCR are as follows:

654-U+D-F：5’-GCTTCTGACACAACTGTGTTC-3’(SEQ ID No.52)；

654-U+D-R：5’-CTTTGCCAAAGTGATGGGCCA-3’(SEQ ID No.53)。

as a result: the results of electrotransformation of sg-U + D (specifically sgRNA-U1 and sgRNA-D1) with large DNA fragments deleted and deleted in the target site of IVS2-654 patient-derived hematopoietic stem cells after electrotransformation of sg-U + D, as compared to a blank control without any RNP introduced, are shown in FIG. 3.

2. EDM-2 and EDM-3 differentiation culture

The method comprises the following steps: and after the gel electrophoresis determines that the target site is successfully deleted, continuously differentiating in the EDM-2 culture medium. The cells in this stage can be greatly amplified, a part of the cells can be frozen in advance for standby, when the EDM-2 differentiation stage is finished, the rest cells are continuously differentiated in the EDM-3, and after the differentiation is finished, RNA is extracted and is reversely converted into cDNA for detection in the following step 3-5.

3. PCR amplification of exons of HBB

The method comprises the following steps: primers 654-exon1-F and 654-exon3-R were used to PCR amplify the differentiated exons to verify whether normal splicing can be achieved after mutation of the pathogenic site. Wherein, the primer sequences are as follows:

654-exon1-F：5’-TGAGGAGAAGTCTGCCGTTAC-3’(SEQ ID No.54)；

654-exon3-R：5’-CACCAGCCACCACTTTCTGA-3’(SEQ ID No.55)。

as a result: compared with a blank control without any RNP, the ratio of the intensity of the abnormal splicing to the normal splicing band of the IVS2-654 patient-derived hematopoietic stem cells is remarkably reduced after sgR-U + D electrotransformation, namely the intron is basically restored to normal splicing after the pathogenic site is edited, wherein the result of electrotransformation of sg-U + D (particularly sgRNA-U1 and sgRNA-D1) is shown in FIG. 4.

4. PCR amplification of CDS region of HBB

The method comprises the following steps: after pathogenic sites of hematopoietic stem cells of a patient are edited and red blood cells are differentiated, an HBB coding region sequence is amplified in a complete cDNA by PCR and Sanger sequencing is carried out; wherein, the primer sequences are as follows:

HBB-CDS-F：5’-ATGGTGCATCTGACTCCTGA-3’(SEQ ID No.56)；

HBB-CDS-R：5’-TTAGTGATACTTGTGGGCCA-3’(SEQ ID No.57)。

as a result: compared with a blank control without any RNP, after sgR-U + D electrotransformation, HBB gene of patient-derived hematopoietic stem cells IVS2-654 recovers normal coding function and is close to healthy cells, and the mutation efficiency can reach 95 percent, wherein the results of the sgR-U + D electrotransformation (specifically sgRNA-U1 and sgRNA-D1) are shown in FIG. 5.

5. qPCR detection

The method comprises the following steps: qPCR analysis is carried out on the variation of globin content in the hemopoietic stem cells after the pathogenic site mutation, wherein the primer sequence used by the qPCR is as follows:

HBA-S：5’-GCCCTGGAGAGGATGTTC-3’(SEQ ID No.58)；

HBA-A：5’-TTCTTGCCGTGGCCCTTA-3’(SEQ ID No.59)；

HBB_e2-e3：5’-TTCAGGCTCCTGGGCAAC-3’(SEQ ID No.60)；

R_HBB_exon3：5’-CACCAGCCACCACTTTCTGA-3’(SEQ ID No.61)。

HBG-S：5’-GGTTATCAATAAGCTCCTAGTCC-3’(SEQ ID No.62)；

HBG-AS：5’-ACAACCAGGAGCCTTCCCA-3’(SEQ ID No.63)。

as a result: the mRNA ratio of HBB to HBA of patient hematopoietic stem cells edited by sgRNA-U and sgRNA-D is close to 60%, which is enough to eliminate erythrocytotoxicity caused by high HBA level and can effectively relieve the symptoms of thalassemia, wherein the result of electrotransformation of sg-U + D (particularly sgRNA-U1 and sgRNA-D1) is shown in FIG. 6.

Example 3 editing of the second intron of HBB in which IVS2-654 sites are located in HUDEP-2 cells

The cells used in this example were HUDEP-2 cells (Human Umbilical Cord Blood depleted Erythoid Progenitor-2, erythroid Progenitor cells Derived from Human Cord Blood).

Editing method

1. Preparation of sgRNA

The sgRNA-U1, sgRNA-U2, sgRNA-U3, sgRNA-D1, sgRNA-D2, sgRNA-D3 and sgRNA-D4 in the table 1 are synthesized by chemical modification.

2. Electrotransformation of sgRNA and Cas9 protein complexes

The method of step 2 in step one of example 2 was followed, except that the following different treatments were provided in total:

blank control: HUDEP-2 cells without any RNP added;

treatment 1: HUDEP-2 cells electroporated with sgRNA-U1 and sgRNA-D1;

and (3) treatment 2: HUDEP-2 cells electroporated with sgRNA-U2 and sgRNA-D2;

and (3) treatment: HUDEP-2 cells electroporated with sgRNA-U2 and sgRNA-D3;

and (4) treatment: HUDEP-2 cells electroporated with sgRNA-U3 and sgRNA-D4.

II, identification result

1. Mutation identification of genomic DNA

The method comprises the following steps: the procedure was followed in step 1 of step two of example 2.

As a result: as shown in FIG. 7, the 1-4 editing results in a DNA deletion of different length in the HBB second intron gene region compared to the blank control without any RNP introduced, and the length of the DNA deletion is the same as expected.

2. EDM-2 and EDM-3 differentiation culture

The procedure was followed in step 2 of step two of example 2.

3. PCR amplification of exons of HBB

The method comprises the following steps: the procedure was followed in step 3 of step two of example 2.

As a result: as shown in FIG. 8, in HUDEP-2 cells, the splicing conditions of the cDNA samples after 1-4 editing treatment, IVS2-654 to the two ends of the HBB second intron gene region, targeted editing and erythrocyte differentiation treatment, were all normal.

The example further verifies that the two sgrnas of the upstream sgRNA and the downstream sgRNA of the pathogenic site are jointly used in the second intron of the HBB for targeted cleavage, so that large-fragment DNA including the pathogenic site is deleted, the expression of the HBB is not affected, and the safety and feasibility are achieved.

Those not described in detail in this specification are within the skill of the art. The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Sequence listing

<110> Shanghai Bodhisae Biotech Co., Ltd, university of east China

<120> sgRNA and application thereof in repairing intron abnormal splicing

<130> JH-CNP200099

<160> 63

<170> PatentIn version 3.5

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Claims

1. A repair method for aberrant splicing of introns due to HBB (β -globin gene) IVS2-654C > T mutation in a cell, said mutation of IVS2-654C > T introducing an additional splice donor site resulting in aberrant splicing of introns, comprising the step of gene editing HBB using CRISPR-Cas9 system to delete said additional splice donor site;

the CRISPR-Cas9 system includes Cas9 and sgrnas that target a target sequence;

the sgRNA includes sgRNA-U and sgRNA-D,

2. The repair method according to claim 1, wherein the sgRNA-U has a targeting sequence of any one or any two or more of the sequences shown in SEQ ID Nos. 1 to 35; preferably, the sequence shown in SEQ ID nos. 1, 2, and/or 3, and more preferably, the sgRNA-U includes a chemical modification of a base.

3. The repair method according to claim 1 or 2, wherein the targeting sequence of the sgRNA-D is any one or any two or more of the sequences shown in SEQ ID Nos. 36 to 51; preferably, the sequence shown as SEQ ID No.36, 37, 38 and/or 39; more preferably, the sgRNA-D comprises a chemical modification of a base.

4. Repair method according to any one of the claims 1-3, characterized in that the sequence of the additional splice donor site is AAGGTAATA.

5. The repair method according to any one of claims 1 to 4, wherein the complex comprising Cas9 and sgRNA-U and the complex of Cas9 and sgRNA-D are introduced into the cell by electrotransformation.

6. The repair method according to any one of claims 1 to 7, wherein the cells are hematopoietic stem cells or erythroid progenitor cells,

preferably, the hematopoietic stem cell is CD34⁺The hematopoietic stem progenitor cell of (1), wherein said erythroid progenitor cell is HUDEP-2.

7. An sgRNA for repairing an intron aberrant splicing caused by an HBB (beta-globin gene) IVS2-654C > T mutation, the sgRNA including sgRNA-U and sgRNA-D,

8. The sgRNA according to claim 7, wherein the targeting sequence of the sgRNA-U is any one or any two or more of the sequences shown in SEQ ID Nos. 1 to 35; preferably, the sequence shown in SEQ ID nos. 1, 2, and/or 3, more preferably, the sgRNA-U comprises a chemical modification of a base;

9. Use of the sgRNA of claim 7 or 8 to repair intron aberrant splicing in a cell caused by a HBB (β -globin gene) IVS2-654C > T mutation.

10. The use according to claim 9, wherein said cells are hematopoietic stem cells or erythroid progenitor cells,