CN113430195A - gRNA molecule of target beta-globin gene, its synthesis method and method for correcting mutation type - Google Patents

Abstract

The application discloses a gRNA molecule of an intron I or an intron II of a targeted beta-globin gene, a synthesis method thereof, a method for constructing a repair system of a targeted beta-globin gene mutation site, a repair system, and a general method for correcting a beta-globin gene mutation type in beta-thalassemia. In particular, the general method is able to correct most types of HBB gene mutations in HSPCs, and the targeted HSPCs function normally in vitro without significant off-target effects.

Description

gRNA molecule of target beta-globin gene, its synthesis method and method for correcting mutation type

Technical Field

The application relates to the field of biotechnology, in particular to a gRNA molecule of an intron I or an intron II of a targeted beta-globin gene, a synthesis method thereof, a method for constructing a repair system of a targeted beta-globin gene mutation site, a repair system, and a general method for correcting a beta-globin gene mutation type in beta-thalassemia.

Background

Beta thalassemia (abbreviated as beta thalassemia) is known to be caused by more than 200 different types of mutations in the beta globin (HBB) gene [1 ]. Typically, HBB pairs with alpha-globulin (HBA) one-to-one to form a tetrameric hemoglobin molecule, and due to insufficient production of HBB, unpaired alpha-globulin chains precipitate, resulting in toxic death of developing red blood cells or red blood cell precursors and insufficient formation of mature Red Blood Cells (RBCs) [2 ]. Ineffective erythropoiesis can lead to anemia, which can lead to higher mortality or shortened life expectancy if not treated in time. Beta thalassemia affects millions of people worldwide, with about 3 per 1,000 newborn infants worldwide suffering from severe beta thalassemia [1, 3 ].

The only effective treatment for beta thalassemia is allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, this approach has limitations in its widespread use due to the lack of immunologically matched donor and graft-versus-host disease [4 ]. Over the last decades, scientists have developed another gene therapy approach to treat beta thalassemia that relies on the genomic inserted lentiviral vectors carrying a functional HBB gene, which is permanently inserted into the genome of autologous Hematopoietic Stem and Progenitor Cells (HSPCs), which will return to the patient's bone marrow after Bone Marrow (BM) transplantation, differentiate into erythrocytes and express high levels of the added HBB gene [5, 6 ]. Although many clinical trials have been conducted to determine the balance between efficacy and risk of gene therapy using beta thalassemia lentiviral vectors, the "semi-random" integration property of lentiviral vectors is always a potential risk [7-9 ]. Current research has enabled precise genome editing through Homology Directed Repair (HDR) of HBB mutations. Unlike viral vector-based gene transfer methods, this method can preserve endogenous promoter and regulate gene expression to mediate spatio-temporal gene expression [10, 11 ].

HDR genome editing is the precise modification of genome nucleotide sequences that requires engineered nucleases to generate DNA Double Strand Breaks (DSBs) at specific genomic sites and DNA donor templates to repair damaged sites through a "copy and paste" mechanism [12 ]. Cas9 nuclease can be programmed under the direction of a single guide rna (grna) to cleave a target locus within the genome with rapid iterations and optimization [13 ]. Recent studies have shown that by combining CRISPR/Cas9 with an exogenous HR donor delivered by a single stranded oligonucleotide (ssODN) [14, 15] or a recombinant adeno-associated viral vector of serotype 6 (rAAV6), Sickle Cell Disease (SCD) point mutation of HBB gene exon 1 in HSPC is effectively targeted for integration, which shows very positive results in vitro, in particular for rAAV6 HDR donors, which can achieve an average HDR efficiency of 29% [16 ].

However, previous studies focused on specific mutations, such as the SCD locus on exon 1 of the HBB gene, and correcting various HBB mutations was more beneficial for future clinical use [17, 18 ]. Therefore, there is a great need to develop a general strategy for correcting most types of HBB mutations by validated CRISPR guide RNA and a DNA donor template for HDR.

Disclosure of Invention

Embodiments of the present application utilize rAAV6 vectors in combination with CRISPR/Cas9 mediated gene editing to successfully effect repair of various types of mutant β -globin genes. The technical solution of the present application uses umbilical cord blood HSPCs from healthy donors and tests rAAV vectors to achieve efficient targeted integration by optimizing the design and delivery parameters of the Ribonucleoprotein (RNP) complex comprising Cas9 protein and modified single-stranded guide RNA, and the rAAV6 donor. Further, HSPC function edited in vitro is assessed by methylcellulose colony assay, CFU assay, and the like. The results show that modified HSPCs show normal multisystem formation in vitro and no off-target mutations, meaning that this strategy demonstrates a general approach to correct most types of HBB gene mutations in beta thalassemia.

Specifically, in a first aspect, the present application provides a gRNA molecule targeting intron I or intron II of the β -globin gene, wherein the gRNA molecule is selected from gRNA I-1, gRNA I-2, gRNA I-3, gRNA II-1, gRNA II-2, and gRNA II-3; the sequence of gRNA I-1 is shown in SEQ ID NO: 1, the sequence of gRNA I-2 is shown as SEQ ID NO: 2, the sequence of gRNA I-3 is shown in SEQ ID NO: 3, the sequence of gRNA II-1 is shown in SEQ ID NO: 4, the sequence of gRNA II-2 is shown in SEQ ID NO: 5, and the sequence of gRNA II-3 is shown in SEQ ID NO: and 6.

Specifically, SEQ ID NO: 1-6 are shown in Table 1 below:

TABLE 1 gRNA molecules targeting intron I or intron II of the beta-globin gene

Preferably, the gRNA molecule is a gRNA II-2 or gRNA II-3 that targets intron II of the β -globin gene, as shown above.

In a second aspect, the present application provides a method of synthesizing a gRNA molecule according to the first aspect, wherein the gRNA molecule template for in vitro transcription is a PCR product obtained from a gRNA vector by using a primer pair, which is a primer T7-F and a primer T7-R, and a high fidelity enzyme; wherein the sequence of the primer T7-F is SEQ ID NO: 7 and the sequence of the primer T7-R is SEQ ID NO: 8.

specifically, SEQ ID NO: 7 is as follows: 5'-GAAATTAATACGACTCACTATA-3', and SEQ ID NO: 8 is as follows: 5'-AAAAAAAGCACCGACTCGGTGCCAC-3' are provided.

In a third aspect, the present application provides a method of constructing a repair system targeting a mutated site of a β -globin gene, comprising the steps of:

step S1: synthesizing Cas9/gRNA RNP;

step S2: performing electrotransfection of the cells;

step S3: after addition of AAV6 donor vector at various MOIs (multiplicity of infection), the electroporated cells were incubated;

step S4: culturing the incubated cells;

wherein the gRNA is selected from gRNA I-1, gRNA I-2, gRNA I-3, gRNA II-1, gRNA II-2 and gRNA II-3;

the sequence of gRNA I-1 is shown in SEQ ID NO: 1, the sequence of gRNA I-2 is shown in SEQ ID NO: 2, the sequence of gRNA I-3 is shown in SEQ ID NO: 3, the sequence of gRNA II-1 is shown in SEQ ID NO: 4, the sequence of gRNA II-2 is shown in SEQ ID NO: 5, and the sequence of gRNA II-3 is shown in SEQ ID NO: and 6.

Preferably, in the above method of constructing a repair system targeting a mutation site of a β -globin gene, Cas9/gRNA RNP is prepared by combining the following genes at room temperature in the ratio of 1: 2.5 molar ratio Cas9 protein was complexed with gRNA.

Preferably, in the above method, the cells in step S2 are hematopoietic stem and progenitor cells (CD34+ HSPC).

Preferably, in the above method, in said step S3, the transfected cells are incubated at 37 ℃.

Preferably, in the above method, in the step S3, the MOI ranges from 1 × 10³To 1X 10⁶More preferably 1X 10⁵。

Preferably, in the above method, in the step S4, at 37 ℃ and 5% CO₂Culturing the incubated cells under the conditions of (1).

Preferably, in the above method, prior to said step S1, in vitro transcription of the gRNA comprises:

cloning the gRNA into a pUC57-T7 vector (Addgene ID: 51306);

sequencing analysis is then performed to select the correct gRNA containing the target site sequence; wherein the gRNA template for in vitro transcription is a PCR product obtained from a gRNA vector by using a primer pair and a high fidelity enzyme, wherein the primer pair is a primer T7-F and a primer T7-R, and the sequence of the primer T7-F is SEQ ID NO: 7 and the sequence of the primer T7-R is SEQ ID NO: 8;

then, the gRNA was transcribed using T7 high yielding RNA synthesis kit and purified using miRNeasy Mini kit;

cas9 expression vectors were linearized using NotI and transcribed using the mmesse mMACHINE SP6 kit to generate capped Cas9 RNA.

Preferably, in the above method, the AAV6 donor vector contains an arm homologous to the β -globin gene with 1.9Kbp on the left and 0.7Kbp on the right.

Further preferably, in the above method, the AAV6 donor vector further comprises SV40 polyA as a STOP, a reporter gene and a promoter for spleen foci forming virus.

Still further preferably, in the above method, the reporter gene is linked downstream of the β -globin gene.

Still further preferably, in the above method, the reporter gene is an EGFP reporter gene or a tNGFR reporter gene.

In a fourth aspect, the present application provides a repair system targeting a mutation site of a β -globin gene constructed by the method of the third aspect.

In a fifth aspect, the present application provides a general method of correcting a mutated type of a β -globin gene in β -thalassemia, wherein the general method uses the repair system of the fourth aspect.

In summary, the present application provides a gRNA molecule targeting intron I or intron II of the β -globin gene, a method of synthesizing the same, a method of constructing a repair system targeting a mutation site of the β -globin gene, a repair system, and a general method of correcting a mutation type of the β -globin gene in β -thalassemia. First, validated grnas with high indel frequency were used as Ribonucleoprotein (RNP) complexes to create DSBs in intron II of the HBB gene. Then, the rAAV6 donor bound to the homology arm was targeted insertion of 3 exons of the HBB gene in the DSB locus. In addition, the technical scheme provided by the application also connects the reporter gene to the downstream of the HBB gene, so that the expression of the reporter gene indicates that the HBB gene is successfully inserted into a genome. By testing this strategy using umbilical cord blood HSPC (CB HSPC) from healthy donors, the inventors found that it enabled efficient targeted insertion and that the edited CB HSPC had normal function compared to the in vitro unmodified cells. Also, whole genome sequencing analysis and off-target results indicate that the modified CB HSPCs exhibit minimal mutation burden and no off-target mutations occur.

In addition, the edited CB HSPCs retain the capacity to transplant after transplantation into immunodeficient non-obese diabetic (NOD) -Severe Combined Immunodeficiency (SCID) IL2rg-/- γ mice (NSI mice); more importantly, the general method provided by the present invention can correct the β -CD41/42 mutation and improve HBB mRNA expression. In addition, the application actually provides an experimental system for screening the small molecular compound based on the reporter gene co-expression, and the screened small molecular compound can improve the HDR efficiency in the HSPC.

Drawings

The drawings are intended for a better understanding of the present solution, and do not limit the present solution.

Figure 1 shows a schematic representation of gene modification of the β -globin gene site (HBB) using CRISPR/Cas9 and rAAV 6; wherein the site-specific DSB is created by CRISPR/Cas9 (red arrow). DSBs use rAAV6 homologous donors as repair templates to promote Homologous Recombination (HR). Light gray frame: a homology arm.

Figure 2 shows CRISPR/Cas 9-mediated targeting of the β -globin gene site (HBB); wherein, fig. 2(a) shows that targeting efficiency of grnas targeting the HBB intron locus within the HBB cell pool was evaluated by TIDE; fig. 2(B) shows Sanger sequencing of target HBB genes by various grnas in HSC cells; figure 2(C) shows that the TIDE evaluated the targeting efficiency of mRNA, RNP (ribonucleoprotein formed by Cas9 protein and gRNA) or rnp.mgrna (chemically modified gRNA ribonucleoprotein) CRISPR system in HSCs cells.

Fig. 3 shows CRISPR/Cas9 and rAAV-mediated gene loci targeting the β -globin gene (HBB). Specifically, fig. 3(a) shows: expression of the EGFP reporter in HSCs transduced with different MOIs of rAAV6 was analyzed by flow cytometry 10 days after delivery of rnp.mgrnaiii-2 and rnp.mgrnaiii-3, respectively, to HSPCs. Fig. 3(B) shows: flow cytometry results of EGFP expression in HSCs transduced with 1E +5MOI rAAV6 following RNP. mg RNAII-2 and RNP. mg RNAII-3 delivery. FIG. 3(C) shows a schematic diagram showing primer sites for detecting HDR by PCR. Fig. 3(D) shows: agarose gel picture of genotype of clones targeted to HBB by rAAV6 after delivery of rnp.mgrnaiii-2 and rnp.mgrnaiii-3 to HSPC. FIG. 3(E) shows HDR efficiency of RNP. mg RNAiII-2 and RNP. mg RNAiII-3 transfected EFGP-positive HSC cells. FIG. 3(F) shows HDR efficiency of all HSC cells transfected with RNP. mg RNAiII-2 and RNP. mg RNAiII-3.

FIG. 4 shows functional analysis of targeted HSPC, where hematopoietic progenitor CFU analysis reveals the ability of lineage restricted progenitors (BFU-E and CFU-GM) and pluripotent progenitors (CFU-GEMM) to form.

Figure 5 is a whole exome sequencing graph of gene-targeted HSPCs showing the number of SNVs revealed by whole exome sequencing in rnp.mgrna-targeted HSPC cells; wherein the grey part represents the background, the red part represents the mutation site and the length represents the genome density.

Detailed Description

Exemplary embodiments of the present application are described below with reference to the drawings, which include various details of embodiments of the present application to facilitate understanding and which are to be considered exemplary only. Accordingly, it will be understood by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, descriptions of well-known functions and constructions are omitted in the following description for clarity and conciseness.

It should be noted that all methods and processes not described in detail in the specification use steps and operations known in the art and are therefore not described in detail herein.

The abbreviated terms used in the examples herein mainly include: HSPC: hematopoietic stem and progenitor cells; CRISPR/Cas: clustered regularly spaced short palindromic repeats; RNP: a ribonucleoprotein complex; rAAV 6: recombinant adeno-associated virus 6; gRNA: a small guide RNA; HDR: homology directional repair; CFU: a colony forming unit; tNGFR: a truncated nerve growth factor receptor; NHEJ: non-homologous end joining; and (3) DSB: double strand breaks; ssODN: a single stranded oligodeoxynucleotide; FACS: fluorescence activated cell sorting.

Cell culture

CD34+ HSPCs from umbilical cord blood and fetal liver were obtained from the obstetrics of the third subsidiary hospital of the university of medical, guangzhou and approved by the ethical committee of that hospital. HSPCs are purified within 24 hours after a predetermined apheresis. First, whole cord blood was mixed with PBS at a ratio of 1: 1(v/v) and then separating the mononuclear fraction by density gradient separation using Ficoll. CD34+ HSPCs were extracted from the mononuclear fraction using a CD34 MicroBead Kit (Miltenyi Biotech, CD34 MicroBead Kit ultra pure, human) according to the manufacturer's protocol. Cells were stained for CD34 using APC anti-human CD34 (clone 563; BD) to test their purity. All CD34+ HSPC were supplemented with SCF (100ng ml)^-1)，TPO(100ng ml^-1) Flt3 ligand (100ng ml)^-1)，IL-6(100ng ml^-1) Stem cell regenerant 1 (0.75. mu.M) and UM171(35nM) in StemBan SFEMII (Stem cell technology). All cells were at 37 ℃ and 5% CO₂Culturing under the conditions of (1).

Production of AAV vector

Cloning of AAV vector plasmids into the ssAAV-MCS plasmid (PackGene Biotech) using Gibson Assembly Mastermix (New England Biolabs)Inverted Terminal Repeats (ITRs) from AAV serotype 2(AAV 2). The HBB AAV6 donor contained an arm homologous to the β -globin locus with 1.9Kbp on the left and 0.7Kbp on the right (see FIG. 1), and SV40 polyA as STOP, reporter gene (EGFP or tNGFR) and spleen foci forming viral promoter. The preparation steps of the AAV6 vector mainly comprise: first, 1X 10 cells were seeded per 15 cm dish prior to transfection⁷293T cells; each 15 cm dish was transfected with 6. mu.g of the ssAAV-MCS plasmid containing the donor, 7.5. mu.g of pAAVcap6 containing the AAV6 cap gene and AAV2 rep gene, and 7.5. mu.g of adenovirus helper gene, respectively, using Polyethyleneimine (PEI). After 72 hours of incubation, the cells were lysed by three freeze-thaw cycles and then incubated with 250U/ml TurboNuclean (Abnova) for 45 minutes. AAV6 particles were purified by density gradient centrifugation at 237,000g of iodixanol at 18 ℃ for 2 h. AAV6 vector was extracted at 60-40% of the iodixanol surface and then exchanged with 5% sorbitol in PBS using a Molecular Weight Cutoff (MWCO) Slide-a Lyzer G2 dialysis cartridge (Thermo Fisher Scientific) according to the manufacturer's instructions. AAV6 vector was titrated using quantitative PCR to measure the number of vector genomes. The vector was stored at-80 ℃.

Design and in vitro transcription of gRNAs

gRNAs targeting either intron I or intron II of the HBB gene (i.e., the β -globin gene) were designed online (http:// crispor. for. net/crispor. py). Cloning the gRNA into a pUC57-T7 vector (Addgene ID: 51306); sequencing analysis is then performed to select the correct gRNA containing the target site sequence. Wherein the gRNA template for in vitro transcription is a PCR product obtained from a gRNA vector by using a primer pair, which is a primer T7-F and a primer T7-R, and a high fidelity enzyme (Takara); wherein the sequence of the primer T7-F is SEQ ID NO: 7 and the sequence of the primer T7-R is SEQ ID NO: 8. The gRNA was then transcribed using the T7 high yield RNA synthesis kit (NEB) and purified using the miRNeasy Mini kit (Qiagen). Cas9 expression vectors were linearized using NotI and transcribed using the mmesse mMACHINE SP6 kit (Ambion) to generate a capped Cas9 RNA. The concentration and quality of the synthesized Cas9 mRNA and gRNA were measured using NanoDrop 2000 and agarose gel (1%) electrophoresis, respectively.

Electroporation and transduction of cells

HBB gRNA was produced by in vitro transcription, and a modified gRNA having 2 '-O-methyl-3' -phosphorothioate modifications at the three terminal nucleotides of the 5 'and 3' ends was synthesized by GENSCRIPT. Cas9 protein was purchased from GENSCRIPT, and prior to electroporation was purified by separation at room temperature at 1: 2.5 molar ratio of Cas9 protein: gRNA Cas9 protein was complexed with gRNA for 10 min to prepare RNP. CD34+ HSPC was electroporated using Lonza Nucleofector 2b (program U-014). The following conditions were used: 5X 10⁶Cells/ml, 300 μ g/ml Cas9 protein with gRNA at 1: 2.5 molar ratio complexation, or 100 μ g/ml synthetic chemically modified grnas with 150/μ g/ml Cas9 mRNA after electroporation, cells were incubated at 37 ℃ for 15 minutes, followed by addition of AAV6 donor vector thereto at various MOIs. The cells were then incubated at 37 ℃ and 5% CO₂And (5) culturing.

TIDE analysis

To determine the gene targeting efficiency of the grnas, PCR products spanning the Cas9-gRNA cleavage site were used for Sanger sequencing and the inde frequency was quantified using the TIDE software (https:// TIDE. The primers used to amplify the PCR fragment of TIDE at the β -globin locus included a forward primer and a reverse primer, wherein the sequence of the forward primer is SEQ ID NO: 9 (5'-GACACCATGGTGCATCTGAC-3'), the sequence of the reverse primer is SEQ ID NO: 10 (5'-TAATGTACTAGGCAGACTGT-3').

Measurement of targeted integration of fluorescent AAV6 donor and methylcellulose CFU assay

The targeted integration rate of the GFP donor was measured by flow cytometry 10 days after electroporation. GFP production by FACS^{Height of}The populations were sorted into 96-well plates containing MethoCult Optimum (Stem cell technology). After 14 days, colonies were counted under an inverted microscope and counted according to colony forming units-erythroid (CFU-E), colony forming unit-granulocyte, monocyte (CFU-GM) and colony forming unit-pluripotent (CFU-GEMM)Morphological features were scored as single blind.

Genotyping of methylcellulose colonies

Colonies formed in methylcellulose were extracted from FACS sorting of single cells and placed in 96-well plates. PBS was first added to the 96-well plate and colonies were mixed with PBS and transferred to V-bottom 96-well plates. Then, the cells were pelleted by centrifugation at 300g for 5 minutes at room temperature, and after removing the supernatant, the cells were resuspended in 250. mu.l of PBS. Subsequently, the cells were again pelleted by centrifugation at 300g for 5 minutes. Finally, the cells were resuspended in 10. mu.l of DNA extraction solution and transferred to PCR plates, which were incubated at 65 ℃ for 60 minutes and then at 95 ℃ for 10 minutes. Integrated alleles were detected using PCR, with the primers used as follows: SEQ ID NO: 11 (F: 5'-TGCCTGGTATGCCTGGGCTT-3') and SEQ ID NO: 12 (R: 5'-CTTCAAGAGGTGGAACAGCT-3').

Whole exome sequencing

Genomic DNA was extracted from WT (wild type) and RNP-mgRNA targeted HSPC cells using a cell genome extraction kit. According to the manufacturer's instruction manual, SeqCap EZ Exome 64M (Roche NimbleGen) and TruSeq DNA sample preparation for capture of Exome and establishment of Exome sequencing library. All sequencing was performed on Illumina NovaSeq using 2 × 150 nucleotide multiplex with paired ends. Quality control was performed using trimmatic (version 0.39) by removing adaptor sequences and low complexity or low quality reads. BWA (0.7.17-r1188 edition) was then used to align pure reads to the hg38 human genome downloaded from UCSCs. Samtools (version 1.9) is used to sort, index the bam file, and Picard (version 2.20.5) is used to mark duplicates. GATK (version 4.1.3.0) was used for the subsequent steps: 1) base quality score recalibration, 2) variant discovery and 3) variant quality score recalibration. Known site resources (e.g., SNPs from the 1000G project) were used for the Variant Quality Score Recalibration (VQSR) program with the parameters (-QD-MQ-MQRankSum-ReadPosRankSum-FS-SOR), and the partial sensitivity threshold was assigned as 90. Variants that pass through the VQSR program are considered true variants. Variant annotation and further filtering were performed using ANNOVAR (24/10/2019) and the genome-wide database (exac03, avsnp 150). Variants tagged to known sites were eliminated. During the downstream analysis, all the variants present in the sample were considered as background noise and eliminated in the further analysis. The mutated genome traces were drawn using circize (version 0.4) and used for other custom visualizations.

Development of a general method for targeting the HBB Gene

To develop a general method for correcting most HBB mutations, the inventors tested the protocol shown in figure 1. First, the inventors designed a method to achieve HDR in intron I or intron II of the HBB locus. Site-specific DSBs are generated from a Ribonucleoprotein (RNP) complex consisting of a gRNA and Cas9 protein, and HDR can be achieved using rAAV6 cognate donors as repair templates. To facilitate the enrichment and tracking of HDR, the inventors further added EGFP or tNGFR reporter genes downstream of the HBB gene.

Optimizing delivery of Cas9/gRNA RNP to hematopoietic stem/progenitor cells

In this application, the inventors selected six grnas with higher scores that target both introns of HBB using a web-based search tool. gRNA II-2 and gRNA II-3 showed the highest targeting efficiency in HSPC pools electroporated with plasmids (fig. 2A, B). Previous studies have shown that the Cas9/gRNA system provided as an RNP complex by electroporation is the most effective method for creating DSBs and stimulating HR in HSPCs [14, 19 ]. Further, previous studies have demonstrated that the modification of the three terminal nucleotides at the 5 'and 3' ends of the gRNA by 2 '-O-methyl-3' -phosphorothioate (MS) significantly improves the ability of the Cas9/gRNA system to induce DSB in HSPC [16, 20 ]. Thus, the present application determines whether modified grnas and RNPs are more effective than mrnas. Cas9-mRNA/gRNA, Cas9/gRNA RNP and Cas9/mgRNA RNP were introduced into HSPCs by electroporation and the cells were harvested four days later, the application found that the modified guide produced a significant increase in the rate of indel at the HBB leader locus when the Cas9/gRNA system was delivered as RNP compared to the unmodified guide (fig. 2C). Most importantly, the targeting efficiency of the HBB leader site can reach 80%.

Optimizing transduction of rAAV6 donors to HSPC to achieve consistently high levels of HDR

Next, the inventors of the present application optimized transduction of rAAV6 donors by titration with MOI or vector genome/cell (vgs/cell) that stimulated the highest rate of HDR in HSPC with appropriate cell viability. After delivery of Cas9/gRNA RNP to cells by electroporation, rAAV6 donors were used at 1 × 10³-1× 10⁶MOI of MOI transduces CB-HSPC. The inventors found that the MOI was 1X 10⁵At this time, the EGFP positivity reached 20% (fig. 3A, B), while the HDR rate did not increase with increasing MOI. Selection of 1X 10⁵The MOI of (2) was used in the following experiment.

Identification of genotype of edited clones at HBB site

To identify the genotype of HSPCs targeting HBBs, the inventors performed single cell methylcellulose cloning of the population. After 10 days of HSPC transduced with rnp.mgrna and rAAV6, EGFP positive cells were sorted and inoculated in methylcellulose medium for 2 weeks. To detect targeted integration in the HBB locus, primers were designed outside the arm of the homolog (fig. 3C). Of EGFP positive cells, about 60% to 85% of cells had targeted integration (fig. 3E, D). Overall, 10% -20% of the transduced HSPC cells were targeted for integration (fig. 3F).

CB after delivery with RNP and rAAV6 donors Functional analysis of HSPC

To see if strategies targeting HBB could affect HSPC function, we performed hematopoietic progenitor CFU assays to show the capacity of lineage-restricted progenitors (BFU-E and CFU-GM) and pluripotent progenitors (CFU-GEMM) to form. The data showed no significant difference between untargeted and targeted EGFP-positive cells (fig. 4). In summary, the data of the examples of the present application show that the strategy to target HBB does not affect HSPC function.

All-out of gene-targeted HSPCSequencing analysis of the set of displays

CRISPR/Cas9 has highly improved gene targeting efficiency, but it also has the potential risk of causing off-target mutations. Since the importance of exon regions can affect the biological function of most cells, the inventors of the present application performed whole exome sequencing. Genomic DNA from sorted EGFP positive cells was used for NGS library construction. The mutations detected in all samples were considered background somatic mutations. No significant mutations were detected in both RNP.mgRNAII-2 and RNP.mgRNAII-3 (FIG. 5).

As described above, the present application has constructed an efficient and versatile repair system targeting the site of mutation of the β -globin gene using Cas9 RNP in combination with rAAV6 homologous donor vectors. Furthermore, the methods of correcting the type of mutation in the β -globin gene provided herein are capable of correcting not only HBB mutations, but also some upstream and downstream mutations in the HBB gene.

In addition, in optimizing targeting efficiency of RNP and rAAV6 donors of the present application, the present application found that the modified synthetic gRNA was significantly higher than the IVT gRNA. Localization efficiencies have been as high as 80%, which is beneficial because modified synthetic grnas are not degraded by cellular immune reactions, and chemical modifications can provide greater stability and protection against the action of exonucleases. This is a conclusion that the inventors and other researchers of the present application have also demonstrated in the prior art [24, 25 ]. Higher indel rates are the basis for high HDR efficiency, which in this application is close to 16%. The present application found that targeted HSPCs retain similar differentiation capacity in vitro compared to untargeted cells. Also, no off-target effects were found associated with the repair system by whole exome sequencing.

In view of the above, the present application actually provides a general method for correcting most types of HBB gene mutations in HSPCs. The targeted HSPCs function normally in vitro with no apparent off-target effects.

The references in the specification of this application are listed below: [1]Raffaella O.β-Thalassemia. genetics inmedicine.2017；19:609-619.[2]Shah FT,Sayani F,Trompeter S,Drasar E, Piga A.Challenges of blood transfusions inβ-thalassemia.Blood reviews.2019； 37:1-13.[3]Cavazzana M,Antoniani C,Miccio A.Gene Therapy for β-Hemoglobinopathies.Molecular therapy:the journal of the American Society of Gene Therapy.2017；25:1142-1154.[4]Jagannath VA,Fedorowicz Z,Al Hajeri A, Sharma A.Hematopoietic stem cell transplantation for people withβ-thalassaemia major.The Cochrane Database of Systematic Reviews.2016；11:CD008708.[5] Brendel C,Williams DA.Current and future gene therapies for hemoglobinopathies. Current opinion in hematology.2020；27:149-154.[6]Lamsfus-Calle A, Daniel-Moreno A,

G,Raju J,Antony J,Handgretinger R et al. Hematopoietic stem cell gene therapy:The optimal use of lentivirus and gene editing approaches.Blood reviews.2019；15:100641.[7]Magrin E,Miccio A,Cavazzana M. Lentiviral and genome-editing strategies for the treatment ofβ-hemoglobinopathies. Blood.2019；134:1203-1213.[8]Thompson AA,Walters MC,Kwiatkowski J,Rasko JEJ,Ribeil JA,Hongeng S et al.Gene Therapy in Patients with Transfusion-Dependent beta-Thalassemia.The New England journal of medicine. 2018；378:1479-1493.[9]Wu X,Li Y,Crise B,Burgess SM.Transcription start regions in the human genome are favored targets for MLV integration.Science.2003； 300:1749-1751.[10]Biffi A.Gene Therapy as a Curative Option for beta-Thalassemia.The New England journal of medicine.2018；378:1551-1552.[11] Dever DP,Porteus MH.The changing landscape of gene editing in hematopoietic stem cells:a step towards Cas9 clinical translation.Current opinion in hematology. 2017；24:481-488.[12]Zhang J,Li XL,Li GH,Chen W,Arakaki C,Botimer GD et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.Genome biology.2017；18:35.[13]Jacinto FV,Link W,Ferreira BI.CRISPR/Cas9-mediated genome editing:From basic research to translational medicine.Journal of cellular and molecular medicine.2020；00:1-13.[14] Dewitt MA,Corn JE,Carroll D.Genome editing via delivery of Cas9 ribonucleoprotein.Methods.2017；121:9-15.[15]Hoban MD,Lumaquin D,Kuo CY, Romero Z,Long J,Ho M et al.CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+cells.Molecular therapy:the journal of the American Society of Gene Therapy.2016；24:1561-1569.[16]Dever DP,Bak RO,Reinisch A, Camarena J,Washington G,Nicolas CE et al.CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells.Nature.2016；539:384-389.[17]Mark AD,Wendy M,Nicolas LB.Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells.Science translational medicine. 2016；8:360ra134.[18]Megan DH,Gregory JC,Matthew CM.Correction of the sickle cell disease mutation in human hematopoietic.Gene Therapy.2015； 29:234-239.[19]Lattanzi A,Meneghini V,Pavani G,Amor F,Ramadier S,Felix T et al.Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements.Molecular therapy:the journal of the American Society of Gene Therapy.2019；27:137-150.[20]Chakrabarti AM,Henser-Brownhill T,Monserrat J,Poetsch AR,Luscombe NM,Scaffidi P. Target-Specific Precision of CRISPR-Mediated Genome Editing.Molecular cell. 2019；73:699-713.[21]Liu Y,Yang Y,Kang X,Lin B,Yu Q,Song B et al.One-Step Biallelic and Scarless Correction of a beta-Thalassemia Mutation in Patient-Specific iPSCs without Drug Selection.Molecular therapy Nucleic acids.2017；6:57-67.[22] Ma Y,Chen W,Zhang X,Yu L,Dong W,Pan S et al.Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein.RNA Biology.2016；13:605-612.[23]Cai L,Bai H,Mahairaki V,Gao Y,He C,Wen Y et al.A Universal Approach to Correct Various HBB Gene Mutations in Human Stem Cells for Gene Therapy of Beta-Thalassemia and Sickle Cell Disease. Stem cells translational medicine.2018；7:87-97.[24]Bak RO,Dever DP,Porteus MH.CRISPR/Cas9 genome editing in human hematopoietic stem cells.Nature protocols.2018；13:358-376.[25]Scott T,Soemardy C,Morris K.Development of a Facile Approach for Generating Chemically Modified CRISPR/Cas9 RNA.Molecular therapy Nucleic acids.2020；19:1176-1185.[26]Vartak SV,Swarup HA, Gopalakrishnan V,Gopinatha VK,Ropars V,Nambiar M et al.Autocyclized and oxidized forms of SCR7 induce cancer cell death by inhibiting nonhomologous DNA end joining in a Ligase IV dependent manner.The FEBS journal.2018； 285:3959-3976.[27]Vartak SV,Raghavan SC.Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing.The FEBS journal. 2015；282:4289-4294.[28]Wagenblast E,Azkanaz M,Smith SA,Shakib L,Mcleod JL,Krivdova G et al.Functional profiling of single CRISPR/Cas9-edited human long-term hematopoietic stem cells.Nature communications.2019；10:10-18。

It is emphasized that the above specific embodiments do not limit the scope of protection of the application. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations and substitutions can be made in accordance with actual needs and other factors. Any modification, equivalent replacement or improvement within the spirit and principle of the present application shall be deemed to fall within the protection scope of the present application.

Claims (10)

1. A gRNA molecule targeting intron I or intron II of a β -globin gene, wherein the gRNA molecule is selected from gRNA I-1, gRNA I-2, gRNA I-3, gRNA II-1, gRNA II-2, and gRNA II-3;

the sequence of gRNA I-1 is shown in SEQ ID NO: 1, the sequence of gRNA I-2 is shown in SEQ ID NO: 2, the sequence of gRNA I-3 is shown in SEQ ID NO: 3, the sequence of gRNA II-1 is shown in SEQ ID NO: 4, the sequence of gRNA II-2 is shown in SEQ ID NO: 5, and the sequence of gRNA II-3 is shown in SEQ ID NO: and 6.

2. The gRNA molecule according to claim 1, characterized in that the gRNA molecule is a gRNA II-2 or a gRNA II-3 that targets intron II of the β -globin gene.

3. A method of synthesizing a gRNA molecule according to claim 1 or 2, characterized in that the gRNA molecule template for in vitro transcription is a PCR product obtained from a gRNA vector by using a primer pair, which is a primer T7-F and a primer T7-R, and a hi-fi enzyme, wherein the sequence of the primer T7-F is SEQ ID NO: 7 and the sequence of the primer T7-R is SEQ ID NO: 8.

4. a method of constructing a repair system targeting a mutated site of a β -globin gene comprising the steps of:

step S1: synthesizing Cas9/gRNA RNP;

step S2: performing electrotransfection of the cells;

step S3: after addition of AAV6 donor vector at various MOIs (multiplicity of infection), the electroporated cells were incubated;

step S4: culturing the incubated cells;

wherein the gRNA is selected from gRNA I-1, gRNA I-2, gRNA I-3, gRNA II-1, gRNA II-2 and gRNA II-3;

the sequence of gRNA I-1 is shown in SEQ ID NO: 1, the sequence of gRNA I-2 is shown in SEQ ID NO: 2, the sequence of gRNA I-3 is shown in SEQ ID NO: 3, the sequence of gRNA II-1 is shown in SEQ ID NO: 4, the sequence of gRNA II-2 is shown in SEQ ID NO: 5, and the sequence of gRNA II-3 is shown in SEQ ID NO: and 6.

5. The method of claim 4, wherein the Cas9/gRNA RNP is generated by contacting at room temperature at 1: 2.5 molar ratio Cas9 protein was complexed with gRNA.

6. The method according to claim 4, wherein the cells in step S2 are hematopoietic stem and progenitor cells (CD34+ HSPC).

7. The method of claim 4, wherein in step S3, the transfected cells are incubated at 37 ℃.

8. The method of claim 4, wherein in the step S3, the MOI is in the range of 1 x 10³To 1X 10⁶Preferably 1X 10⁵。

9. The method of claim 4, wherein in the step S4, the temperature is 37 ℃ and the CO content is 5%₂Culturing the incubated cells under the conditions of (1).

10. The method of claim 4, wherein prior to said step S1, in vitro transcription of gRNAs comprises:

cloning the gRNA into a pUC57-T7 vector (Addgene ID: 51306);

sequencing analysis is then performed to select the correct gRNA containing the target site sequence; wherein the gRNA template for in vitro transcription is a PCR product obtained from a gRNA vector by using a primer pair and a high fidelity enzyme, wherein the primer pair is a primer T7-F and a primer T7-R, and the sequence of the primer T7-F is SEQ ID NO: 7 and the sequence of the primer T7-R is SEQ ID NO: 8;

then, the gRNA was transcribed using T7 high yielding RNA synthesis kit and purified using miRNeasy Mini kit;

cas9 expression vectors were linearized using NotI and transcribed using the mmesse mMACHINE SP6 kit to generate capped Cas9 RNA.

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