WO2022147759A1 - Grna molecule targeting intron i or intron ii of hbb gene, synthetic method thereof, and method to correct types of hbb gene mutations - Google Patents

Grna molecule targeting intron i or intron ii of hbb gene, synthetic method thereof, and method to correct types of hbb gene mutations Download PDF

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WO2022147759A1
WO2022147759A1 PCT/CN2021/070839 CN2021070839W WO2022147759A1 WO 2022147759 A1 WO2022147759 A1 WO 2022147759A1 CN 2021070839 W CN2021070839 W CN 2021070839W WO 2022147759 A1 WO2022147759 A1 WO 2022147759A1
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grna
sequence
seq
cells
cas9
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PCT/CN2021/070839
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Sheng ZENG
Xinping Chen
Zongfan YANG
Meimei FENG
Ye Wang
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Susheng Biotech (Hainan) Co., Ltd.
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Priority to CN202110493308.4A priority patent/CN113430195A/en
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Definitions

  • the present application relates to the field of biotechnology, and in particular, to a gRNA molecule targeting Intron I or Intron II of ⁇ -globin gene, a synthetic method thereof, a method for constructing a repair system targeting ⁇ -globin gene mutation sites, a repair system, and a universal method to correct types of ⁇ -globin gene mutations in ⁇ -thalassemia.
  • ⁇ -thalassemia (abbreviated as ⁇ -thal) is caused by over 200 different types of mutations in the ⁇ -globin (HBB) gene [1] .
  • HBB ⁇ -globin
  • HBA ⁇ -globin
  • unpaired ⁇ -globin chains precipitate, thereby causing toxic death to the developing erythrocyte or erythrocyte precursor and leading to the insufficient formation of mature red blood cells (RBCs) [2] .
  • RBCs mature red blood cells
  • Ineffective erythropoiesis leads to anemia, and severe anemia can cause a high level of mortality or shortened life expectancy if left untreated.
  • ⁇ -thal affects millions of people worldwide, and approximately 3 of 1,000 new births worldwide are affected with a severe form of ⁇ -thal [1, 3] .
  • allo-HSCT allogeneic hematopoietic stem cell transplantation
  • HDR homology-directed repair
  • HDR genome editing is the precise modification of the nucleotide sequence of the genome, it requires engineered nucleases to create DNA double-strand breaks (DSBs) at a specific genomic site and a DNA donor template to repair the damaged site through a “copy and paste” mechanism [12] .
  • the Cas9 nuclease guided by a single guide RNA (gRNA) , can be programmed to cut a target locus within the genome with rapid iteration and optimization [13] .
  • gRNA single guide RNA
  • SCD sickle cell disease
  • ssODNs single-stranded oligonucleotides
  • rAAV6 recombinant adeno-associated viral vectors of serotype 6
  • Embodiments of the present application successfully utilize rAAV6 vector to achieve repaired various types of mutations ⁇ -globin gene combined with the CRISPR/Cas9 mediated gene editing.
  • Technical solutions in the present application use cord blood-derived HSPCs from health donors, and test rAAV vectors to achieve highly efficient targeted integration by optimizing design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and modified single guide RNA, together with a rAAV6 donor.
  • RNP ribonucleoprotein
  • the edited HSPCs function in vitro is assessed by methylcellulose colonies assay, CFU assay, etc. Results shows that corrected HSPCs exhibit normal multilineage formation in virto and without off-target mutagenesis, which means this strategy demonstrates a universal approach to correct most types of HBB gene mutations in ⁇ -thal.
  • the present application provides a gRNA molecule targeting Intron I or Intron II of ⁇ -globin gene, wherein the gRNA molecule is selected from the group consisting of 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 as 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 as SEQ ID NO: 3, the sequence of gRNA II-1 is shown as SEQ ID NO: 4, the sequence of gRNA II-2 is shown as SEQ ID NO: 5, and the sequence of gRNA II-3 is shown as SEQ ID NO: 6.
  • SEQ ID NOs: 1-6 are shown in Table 1:
  • the gRNA molecule is gRNA II-2 or gRNA II-3 targeting Intron II of ⁇ -globin gene, as shown above.
  • the present application provides a synthetic method of the gRNA molecule according to the first aspect, wherein templates of the gRNA molecule for in vitro transcription are PCR products obtained from gRNA vectors by using primer pairs, which are T7-F and T7-R, with a high-fidelity enzyme; wherein the sequence of the T7-F is SEQ ID NO: 7, and the sequence of the T7-R is SEQ ID NO: 8.
  • SEQ ID NO: 7 is shown as follows: 5′-GAAATTAATACGACTCACTATA-3′
  • SEQ ID NO: 8 is shown as follows: 5′-AAAAAAAGCACCGACTCGGTGCCAC-3′.
  • the present application provides a method for constructing a repair system targeting ⁇ -globin gene mutation sites, comprising steps of:
  • S1 synthesizing Cas9/gRNA RNP
  • the gRNA is selected from the group consisting of gRNA I-1, gRNA I-2, gRNA I-3, gRNA II-1, gRNA II-2 and gRNA II-3;
  • sequence of gRNA I-1 is shown as SEQ ID NO: 1
  • sequence of gRNA I-2 is shown as SEQ ID NO: 2
  • sequence of gRNA I-3 is shown as SEQ ID NO: 3
  • sequence of gRNA II-1 is shown as SEQ ID NO: 4
  • sequence of gRNA II-2 is shown as SEQ ID NO: 5
  • sequence of gRNA II-3 is shown as SEQ ID NO: 6.
  • the Cas9/gRNA RNP is made by complexing Cas9 protein with the gRNA at a molar ratio of 1: 2.5 at room temperature.
  • the cells in S2 is CD34+ HSPCs (Hematopoietic Stem and Progenitor Cells) .
  • the electroporated cells are incubated at 37 °C in S3.
  • the MOI ranges from 1 ⁇ 10 3 to 1 ⁇ 10 6 , more preferably 1 ⁇ 10 5 , in S3.
  • the incubated cells are cultured at 37 °Cand 5%CO 2 in S4.
  • in vitro transcription of the gRNA comprises:
  • the gRNAs are cloned into the pUC57-T7 vector (Addgene ID: 51306) ;
  • templates of the gRNA for in vitro transcription are PCR products obtained from gRNA vectors by using primer pairs, which are T7-F and T7-R, with a high-fidelity enzyme; wherein the sequence of the T7-F is SEQ ID NO: 7, and the sequence of the T7-R is SEQ ID NO: 8;
  • the gRNA is transcribed using a T7 High Yield RNA Synthesis Kit and purified using a miRNeasy Mini Kit;
  • a Cas9 expression vector is linearized using NotI and transcribed using the mMESSAGE mMACHINE SP6 Kit to produce capped Cas9 RNA.
  • the AAV6 donor vectors contain arms homologous to the ⁇ -globin gene of 1.9Kbp on the left side and 0.7Kbp on the right side.
  • the AAV6 donor vectors further contain SV40 polyA as STOP, a reporter gene, and spleen focus forming virus promoters.
  • the reporter gene is linked downstream to the ⁇ -globin gene.
  • the reporter gene is EGFP reporter gene or tNGFR reporter gene.
  • the present application provides a repair system targeting ⁇ -globin gene mutation sites constructed by the method according to the third aspect.
  • the present application provides a universal method to correct types of ⁇ -globin gene mutations in ⁇ -thalassemia, wherein the universal method uses the repair system according to the fourth aspect.
  • the present application provides a gRNA molecule targeting Intron I or Intron II of ⁇ -globin gene, a synthetic method thereof, a method for constructing a repair system targeting ⁇ -globin gene mutation sites, a repair system, and a universal method to correct types of ⁇ -globin gene mutations in ⁇ -thalassemia.
  • a validated gRNA with a high indel frequency is used as a ribonucleoprotein (RNP) complex to create a DSB in Intron II of the HBB gene.
  • RNP ribonucleoprotein
  • the rAAV6 donor combined with homologous arms is targeted insertion of 3 exons of the HBB gene in the DSB locus.
  • the present application also links a reporter gene downstream to the HBB gene so that the expression of the reporter gene is indicative of successful insertion of the HBB gene into the genome.
  • CB HSPCs cord blood-derived HSPCs
  • the edited CB HSPCs retain the ability to engraft when transplanted into immunodeficient nonobese diabetic (NOD) -severe combined immunodeficiency (SCID) IL2rg-/- (gamma) mice (NSI mice) ; more importantly, the universal method can correct the ⁇ -CD41/42 mutation and improve HBB mRNA expression.
  • NOD nonobese diabetic
  • SCID severe combined immunodeficiency
  • NBI mice immunodeficient nonobese diabetic mice
  • the universal method can correct the ⁇ -CD41/42 mutation and improve HBB mRNA expression.
  • the present application provides an experimental system to screen the small-molecule compounds to improve HDR efficiency in HSPCs based on the co-expression of the reporter gene.
  • FIG. 1 shows a schematic diagram of gene correction of ⁇ -globin gene locus (HBB) using CRISPR/Cas9 and rAAV6; wherein, site-specific DSBs are created by CRISPR/Cas9 (red arrow) .
  • a DSB stimulates homologous recombination (HR) using the rAAV6 homologous donor as a repair template.
  • Light gray boxes homology arms.
  • FIG. 2 shows CRISPR/Cas9 mediated targeting of ⁇ -globin gene locus (HBB) ; wherein, (A) shows the targeting efficiency of gRNAs targeted to the HBB intron locus in pools of HSC cells was assessed by TIDE; (B) shows sanger sequence of the targeted HBB gene by various gRNAs in HSCs cells; (C) shows the targeting efficiency of mRNA, RNP (Cas9 protein forms ribonucleoprotein with gRNA) or RNP. mgRNA (chemical modified-gRNA ribonucleoprotein ) CRISPR system in HSCs cells, which were assessed by TIDE.
  • HBB ⁇ -globin gene locus
  • FIG. 3 shows CRISPR/Cas9 and rAAV mediated targeting ⁇ -globin gene (HBB) locus.
  • FIG. 3 (A) shows the expression of EGFP reporter gene in HSCs transduced with different MOIs of rAAV6 was analyzed by flow cytometry 10 days after the delivery of RNP. mgRNAiII-2 and RNP. mgRNAiII-3 into HSPCs.
  • FIG. 3 (B) shows flow cytometry results of the EGFP expression in HSCs transduced with 1E+5 MOI rAAV6 after the delivery of RNP. mgRNAII-2 and RNP. mgRNAII-3.
  • FIG. 3 (A) shows the expression of EGFP reporter gene in HSCs transduced with different MOIs of rAAV6 was analyzed by flow cytometry 10 days after the delivery of RNP. mgRNAiII-2 and RNP. mgRNAiII-3 into HSPCs.
  • FIG. 3 (B) shows flow cyto
  • FIG. 3 (C) shows a schematic diagram indicating the primer site for the detection of HDR by PCR.
  • FIG. 3 (D) shows agarose gel images of the genotypes of clones targeted at the HBB by rAAV6 after the delivery of RNP. mgRNAiII-2 and RNP. mgRNAiII-3 into HSPCs.
  • FIG. 3 (E) shows the HDR efficiency of EFGP positive HSCs cells transfected with RNP. mgRNAiII-2 and RNP. mgRNAiII-3.
  • FIG. 3 (F) shows the HDR efficiency of all HSCs cells transfected with RNP. mgRNAiII-2 and RNP. mgRNAiII-3.
  • FIG. 4 shows functional analysis of targeted HSPCs, wherein hematopoietic progenitor CFU assay reveals the forming ability of lineage-restricted progenitors (BFU-E, and CFU-GM) and multipotent progenitors (CFU-GEMM) .
  • FIG. 5 is a graph of whole exome sequencing of the gene-targeted HSPCs, the graph shows the number of SNVs revealed by whole-exome sequencing in HSPCs cells targeted by RNP. mgRNA; the gray corresponds to background and the red corresponds to mutation sites, the lengths represent genomic density.
  • HSPCs Hematopoietic Stem and Progenitor Cells
  • CRISPR/Cas Clustered regu larly interspaced short palindromic repeats
  • RNP Ribonucleoprotein complex
  • rAAV6 recombinant adeno-associated virus6
  • gRNA small guide RNA
  • HDR homology-directed repair
  • CFU Colony-Forming Unit
  • tNGFR the truncated nerve growth factor receptor
  • NHEJ non-homology end joining
  • DSB double-strand break.
  • ssODNs single-strand oligodeoxynucleotides
  • FACS Fluorescent-activated cell sorting.
  • CD34+ HSPCs from cord blood and fetal liver were obtained from the Department of Obstetrics and Gynecology at The Third affiliated Hospital of Guangzhou Medical University, which was approved by the ethics committee of the hospital. HSPCs were purified within 24 h of scheduled apheresis. Briefly, whole cord blood was mixed with PBS in a proportion of 1: 1 (v/v) , and then the mononuclear fraction was separated by density gradient separation using Ficoll. CD34+ HSPCs were extracted from the mononuclear fraction using a CD34 Microbeads Kit (Miltenyi Biotech, CD34 MicroBead Kit UltraPure, human) according to the manufacturers’ protocol.
  • CD34 Microbeads Kit Miltenyi Biotech, CD34 MicroBead Kit UltraPure, human
  • CD34+ HSPCs were cultured in StemSpan SFEMII (StemCell Technologies) supplemented with SCF (100 ng ml -1 ) , TPO (100 ng ml -1 ) , Flt3 ligand (100 ng ml -1 ) , IL-6 (100 ng ml -1 ) , Stem Regenin1 (0.75 ⁇ M) and UM171 (35 nM) . Cells were cultured at 37 °C and 5%CO 2 .
  • AAV vector plasmids were cloned in the ssAAV-MCS plasmid (PackGene Biotech) , containing inverted terminal repeats (ITRs) from AAV serotype 2 (AAV2) using Gibson Assembly Mastermix (New England Biolabs) .
  • the HBB AAV6 donors contained arms homologous to the beta-globin locus of 1.9Kbp on the left side and 0.7Kbp on the right side (FIG. 1) , the donor also contained SV40 polyA as STOP, a report gene (EGFP or tNGFR) , and spleen focus forming virus promoters.
  • AAV6 vectors were produced as follows: briefly, 1X10 7 293T cells were seeded per 15-cm dish before transfection; each 15-cm dish was transfected with 6 ⁇ g of ssAAV-MCS plasmid containing the donor, 7.5 ⁇ g of pAAVcap6 containing the AAV6 cap genes and AAV2 rep genes and 7.5 ⁇ g of adenovirus helper genes using polyethylenimine (PEI) . After incubating for 72 h, cells were lysed by three freeze-thaw cycles and then incubated with TurboNuclease (Abnova) at 250 U/ml for 45 min.
  • PEI polyethylenimine
  • AAV6 particles were purified by iodixanol density gradient centrifugation at 237,000 g for 2 h at 18 °C.
  • AAV6 vectors were extracted at the 60-40%iodixanol interface and then exchanged in PBS with 5%sorbitol using either a molecular weight cut off (MWCO) Slide-A Lyzer G2 dialysis cassette (Thermo Fisher Scientific) following the manufacturer’s instructions.
  • MWCO molecular weight cut off
  • Slide-A Lyzer G2 dialysis cassette Thermo Fisher Scientific
  • the gRNAs targeting Intron I or Intron II of the HBB gene were designed online (http: //crispor. tefor. net/crispor. py) .
  • the gRNAs were cloned into the pUC57-T7 vector (Addgene ID: 51306) ; subsequent sequencing analysis was performed to select the correct gRNA that contained the target site sequence.
  • Templates of the gRNA for in vitro transcription are PCR products obtained from gRNA vectors by using primer pairs, which are T7-F and T7-R, with a high-fidelity enzyme (Takara) ; wherein the sequence of the T7-F is SEQ ID NO: 7, and the sequence of the T7-R is SEQ ID NO: 8.
  • the gRNAs were transcribed using a T7 High Yield RNA Synthesis Kit (NEB) and purified using a miRNeasy Mini Kit (Qiagen) .
  • the Cas9 expression vector was linearized using NotI and transcribed using the mMESSAGE mMACHINE SP6 Kit (Ambion) to produce 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.
  • the HBB gRNAs were produced from in vitro transcription, and the modified gRNA, which had 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 RNP was made by complexing the Cas9 protein with gRNA at a molar ratio of 1: 2.5 (Cas9 protein: gRNA) at room temperature for 10 min before electroporation.
  • CD34+ HSPCs were electroporated using Lonza Nucleofector 2b (program U-014) .
  • the following conditions were used: 5 ⁇ 10 6 cells/ml, 300 ⁇ g/ml Cas9 protein complexed with gRNA at 1: 2.5 molar ratio, or 100 ⁇ g/ml synthetic chemically modified gRNA with 150/ ⁇ g/ml Cas9 mRNA following electroporation, cells were incubated for 15 min at 37 °C after which they were added AAV6 donor vector at various MOI. Then cells were cultured at 37 °C and 5%CO 2 .
  • Primers used for amplifying PCR fragments for TIDE at the beta-globin locus comprise 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’) .
  • Rates of targeted integration of GFP donors were measured by flow cytometry 10 days after electroporation.
  • the GFP high populations were sorted into 96-well plates containing MethoCult Optimum (Stem Cell Technologies) by FACS. After 14 days, colonies were counted under an inverted microscope and scored in a blinded fashion based on morphological features of colony forming units-erythroid (CFU-E) , colony forming unit-granulocytes, monocytes (CFU-GM) , and colony forming unit-multipotential cells (CFU-GEMM) .
  • CFU-E colony forming units-erythroid
  • CFU-GM colony forming unit-granulocytes
  • CFU-GEMM colony forming unit-multipotential cells
  • Colonies formed in methylcellulose were extracted from FACS sorting of single cells into 96-well plates. Briefly, PBS was added to the 96-well plates, and the colonies were mixed with PBS and transferred to a V-bottomed 96-well plate. Then, the cells were pelleted by centrifugation at 300 g for 5 min at room temperature, and the cells were resuspended in 250 ⁇ l of PBS after removing the supernatant. Afterwards, the cells were pelleted by centrifugation at 300 g for 5 min again. Finally, cells were resuspended in 10 ⁇ l DNA Extraction Solution and transferred to PCR plates, which were incubated at 65 °C for 60 min followed by 95 °C for 10 min.
  • PCR was used to detect the integrated alleles, primers are shown as SEQ ID NO: 11 (F: 5’-TGCCTGGTATGCCTGGGCTT-3’) and SEQ ID NO: 12 (R: 5’-CTTCAAGAGGTGGAACAGCT-3’) .
  • Genomic DNA was extracted from WT (Wild Type) and RNP-mgRNA targeted HSPCs cells using the Cell Genome Extraction kit.
  • SeqCap EZ Exome 64M (Roche NimbleGen) and a TruSeq DNA sample preparation were used to capture the exome and establish the exome sequencing library according to the manufacturers’ instruction manuals. All sequencing was performed on an Illumina NovaSeq with a paired end 2x 150-nucleotide multiplex. Quality control was performed by removing adapter sequences and reads with low complexity or of low quality using Trimmoatic (version 0.39) . Subsequently, BWA (version 0.7.17-r1188) was used to align clean reads to hg38 human genome downloaded from UCSC.
  • VQSR Variant Quality Score Recalibration
  • Variant annotation and further filtration were carried out using ANNOVAR (version 2019 Oct 24) and whole-genome databases (exac03, avsnp150) . Variants annotated to known sites were eliminated. During the downstream analysis, variants that appeared in all samples were considered as background noise, and were eliminated in further analysis. Genomic tracks of mutations were drawn using Circlize (version 0.4. ) was used for other customized visualizations.
  • Site-specific DSB was created by the ribonucleoprotein (RNP) complex consisting of gRNA and Cas9 protein, and HDR was achieved using an rAAV6 homologous donor as a repair template.
  • RNP ribonucleoprotein
  • inventors further added an EGFP or tNGFR reporter gene downstream of the HBB gene.
  • gRNA II-2 and gRNA II-3 showed the highest on-target efficiency in pools of HSPCs electroporated with a plasmid (FIG. 2A, B) .
  • Previous studies have clarified that the Cas9/gRNA system delivered as an RNP complex by electroporation is the most effective method for creating DSBs and stimulating HR in HSPCs [14, 19] .
  • the present application found a significant indel ratio increase generated at the HBB intro locus when the Cas9/gRNA system was delivered as an RNP with modified guides versus unmodified guides (FIG. 2C) . Above all, the targeting efficiency the HBB intro locus can reach to 80%.
  • the inventors next optimized rAAV6 donor transduction by titering the MOI or vector genomes/cell (vgs/cell) that stimulated the highest rates of HDR in HSPCs with an appropriate cell survival rate.
  • CB-HSPCs were transduced with the rAAV6 donor at MOIs ranging from 1 ⁇ 10 3 -1 ⁇ 10 6 MOI.
  • the inventors found that the rates of EGFP positive reached to 20%at an MOI of 1 ⁇ 10 5 (FIG. 3A, B) , and the rates of HDR were not increased with increasing MOI.
  • An MOI of 1 ⁇ 10 5 for the following experiments was chosen.
  • HBB-targeted HSPCs In order to identify the genotype of HBB-targeted HSPCs, the inventors performed single-cell methylcellulose cloning of populations. After 10 days HSPCs transduced with RNP. mgRNA and rAAV6, EGFP positive cells were sorted and seeded in methylcellulose medium for 2 weeks. For detecting the targeted integration in HBB locus, primers were designed outside of the homologues arms (FIG. 3C) . In the EGFP positive cells, about 60%-85%cells had targeted integration (FIG. 3E, D) . Overall, 10%-20%of the trans ducted HSPCs cells were targeted integration (FIG. 3F) .
  • the inventors performed a hematopoietic progenitor CFU assay to show the forming ability of lineage-restricted progenitors (BFU-E, and CFU-GM) and multipotent progenitors (CFU-GEMM) .
  • BFU-E, and CFU-GM lineage-restricted progenitors
  • CFU-GEMM multipotent progenitors
  • CRISPR/Cas9 has highly improved gene targeting efficiency, but it has a potential risk of causing off-target mutagenesis. Due to the importance of exon regions, which affect most cell biological functions, the inventors performed the whole exome sequencing. Genomic DNA from sorted EGFP positive cells were used for NGS library construct. Mutations detected in all samples were considered as background somatic mutations. There was no significant mutations sited were detected in both RNP. mgRNAII-2 and RNP. mgRNAII-3 (FIG. 5) .
  • the present application has established an efficient and universal repair system targeting ⁇ -globin gene mutation sites using the Cas9 RNP in combination with rAAV6 homologous donor vectors. Furthermore, the method to correct types of ⁇ -globin gene mutations provided by the present application enables correction of not only HBB mutations but also some upstream and downstream mutations in the HBB gene.
  • the present application finds that the modified synthetic gRNA is significantly higher than IVT gRNA.
  • the targeting efficiency were archived to 80%, which is beneficial because the modified synthetic gRNA is not degraded by the cellular immune response, and chemical modification provides greater stability and protection from exonucleases. This is a finding that our group and others have also demonstrated in the prior art [24, 25] .
  • a higher indel ratio is the basis of high HDR efficiency, and it is nearly 16%HDR efficiency in the present application.
  • the present application finds that the targeted HSPCs hold similar differential ability in vitro compare with untargeted cells. And there is no off-target effect that is related to the repair system by whole exome sequencing.
  • the present application provides a universal strategy to correct most types of HBB gene mutations in HSPCs.
  • the targeted HSPCs have normal function in vitro and no significant off-target effect.

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Abstract

A gRNA molecule targeting Intron I or Intron II of β-globin gene, a synthetic method thereof, a method for constructing a repair system targeting β-globin gene mutation sites, a repair system, and a universal method to correct types of β-globin gene mutations in β-thalassemia are provided. Especially, the universal method can correct most types of HBB gene mutations in HSPCs, and the targeted HSPCs have normal function in vitro and no significant off-target effect.

Description

GRNA MOLECULE TARGETING INTRON I OR INTRON II OF HBB GENE, SYNTHETIC METHOD THEREOF, AND METHOD TO CORRECT TYPES OF HBB GENE MUTATIONS TECHNICAL FIELD
The present application relates to the field of biotechnology, and in particular, to a gRNA molecule targeting Intron I or Intron II of β-globin gene, a synthetic method thereof, a method for constructing a repair system targeting β-globin gene mutation sites, a repair system, and a universal method to correct types of β-globin gene mutations in β-thalassemia.
BACKGROUND
It is well known that β-thalassemia (abbreviated as β-thal) is caused by over 200 different types of mutations in the β-globin (HBB) gene [1] . Normally, HBB pairs with α-globin (HBA) in a one-to-one ratio to form the tetrameric hemoglobin molecule, and with the insufficient production of HBB, unpaired α-globin chains precipitate, thereby causing toxic death to the developing erythrocyte or erythrocyte precursor and leading to the insufficient formation of mature red blood cells (RBCs) [2] . Ineffective erythropoiesis leads to anemia, and severe anemia can cause a high level of mortality or shortened life expectancy if left untreated. β-thal affects millions of people worldwide, and approximately 3 of 1,000 new births worldwide are affected with a severe form of β-thal [1, 3] .
The only curative treatment for β-thal is allogeneic hematopoietic stem cell transplantation (allo-HSCT) . However, it is limited because of a lack of immunologically matched donors and graft-versus-host disease [4] . In recent decades, scientists have developed an alternative approach of gene therapy for treating β-thal and this approach relies on genome-inserting lentiviral vectors that carry the functional HBB gene, permanently inserting them into the genome of autologous hematopoietic Stem and Progenitor Cells (HSPCs) ,  which will home into the patient’s marrow after bone marrow (BM) transplantation, differentiate into erythrocytes and express a high level of the added HBB gene [5, 6] . Although many clinical trials have been implemented to determine the balance of efficacy and risks for gene therapy with lentiviral vectors for β-thal, the "semi-random" integration nature of the lentiviral vector is always a potential risk. [7-9] . Currently, studies have moved to achieve precise genome editing through homology-directed repair (HDR) of an HBB mutation. Unlike viral-vector-based gene transfer methods, it can preserve endogenous promoters and regulatory gene expression to mediate spatiotemporal gene expression [10, 11] .
HDR genome editing is the precise modification of the nucleotide sequence of the genome, it requires engineered nucleases to create DNA double-strand breaks (DSBs) at a specific genomic site and a DNA donor template to repair the damaged site through a “copy and paste” mechanism [12] . The Cas9 nuclease, guided by a single guide RNA (gRNA) , can be programmed to cut a target locus within the genome with rapid iteration and optimization [13] . Recent studies have demonstrated efficient targeted integration of sickle cell disease (SCD) point mutations in exon 1 of the HBB gene in HSPCs by combining CRISPR/Cas9 with exogenous HR donors delivered via single-stranded oligonucleotides (ssODNs) [14, 15] or recombinant adeno-associated viral vectors of serotype 6 (rAAV6) , which show very positive results in vitro, especially for the rAAV6 HDR donor, which can achieve an average of 29%HDR efficiency [16] .
However, a previous study focused on a specific mutation, such as the SCD locus in exon 1 of the HBB gene and correcting various HBB mutations is more beneficial for future clinical applications [17, 18] . It is highly desirable to develop a universal strategy to correct most types of HBB mutations by validated CRISPR guide RNA and one DNA donor template for HDR.
SUMMARY
Embodiments of the present application successfully utilize rAAV6 vector to achieve repaired various types of mutations β-globin gene combined with the CRISPR/Cas9 mediated gene editing. Technical solutions in the present application use cord blood-derived  HSPCs from health donors, and test rAAV vectors to achieve highly efficient targeted integration by optimizing design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and modified single guide RNA, together with a rAAV6 donor. Moreover, the edited HSPCs function in vitro is assessed by methylcellulose colonies assay, CFU assay, etc. Results shows that corrected HSPCs exhibit normal multilineage formation in virto and without off-target mutagenesis, which means this strategy demonstrates a universal approach to correct most types of HBB gene mutations in β-thal.
Specifically, in a first aspect, the present application provides a gRNA molecule targeting Intron I or Intron II of β-globin gene, wherein the gRNA molecule is selected from the group consisting of 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 as 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 as SEQ ID NO: 3, the sequence of gRNA II-1 is shown as SEQ ID NO: 4, the sequence of gRNA II-2 is shown as SEQ ID NO: 5, and the sequence of gRNA II-3 is shown as SEQ ID NO: 6.
Particularly, SEQ ID NOs: 1-6 are shown in Table 1:
Table 1 gRNA molecule targeting Intron I or Intron II of β-globin gene
Figure PCTCN2021070839-appb-000001
Preferably, the gRNA molecule is gRNA II-2 or gRNA II-3 targeting Intron II of β-globin gene, as shown above.
In a second aspect, the present application provides a synthetic method of the gRNA molecule according to the first aspect, wherein templates of the gRNA molecule for in vitro transcription are PCR products obtained from gRNA vectors by using primer pairs,  which are T7-F and T7-R, with a high-fidelity enzyme; wherein the sequence of the T7-F is SEQ ID NO: 7, and the sequence of the T7-R is SEQ ID NO: 8.
Specifically, SEQ ID NO: 7 is shown as follows: 5′-GAAATTAATACGACTCACTATA-3′, and SEQ ID NO: 8 is shown as follows: 5′-AAAAAAAGCACCGACTCGGTGCCAC-3′.
In a third aspect, the present application provides a method for constructing a repair system targeting β-globin gene mutation sites, comprising steps of:
S1: synthesizing Cas9/gRNA RNP;
S2: performing electroporation of cells;
S3: incubating electroporated cells after they are added AAV6 donor vectors at various MOI (Multiplicity of Infection) ;
S4: culturing incubated cells;
wherein, the gRNA is selected from the group consisting of 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 as 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 as SEQ ID NO: 3, the sequence of gRNA II-1 is shown as SEQ ID NO: 4, the sequence of gRNA II-2 is shown as SEQ ID NO: 5, and the sequence of gRNA II-3 is shown as SEQ ID NO: 6.
Preferably, in the aforementioned method for constructing a repair system targeting β-globin gene mutation sites, the Cas9/gRNA RNP is made by complexing Cas9 protein with the gRNA at a molar ratio of 1: 2.5 at room temperature.
Preferably, in the aforementioned method, the cells in S2 is CD34+ HSPCs (Hematopoietic Stem and Progenitor Cells) .
Preferably, in the aforementioned method, the electroporated cells are incubated at 37 ℃ in S3.
Preferably, in the aforementioned method, the MOI ranges from 1×10 3 to 1×10 6, more preferably 1×10 5, in S3.
Preferably, in the aforementioned method, the incubated cells are cultured at 37 ℃and 5%CO 2 in S4.
Preferably, in the aforementioned method, before S1, in vitro transcription of the  gRNA comprises:
the gRNAs are cloned into the pUC57-T7 vector (Addgene ID: 51306) ;
subsequent sequencing analysis is performed to select the correct gRNA that contained target site sequence; wherein, templates of the gRNA for in vitro transcription are PCR products obtained from gRNA vectors by using primer pairs, which are T7-F and T7-R, with a high-fidelity enzyme; wherein the sequence of the T7-F is SEQ ID NO: 7, and the sequence of the T7-R is SEQ ID NO: 8;
and then, the gRNA is transcribed using a T7 High Yield RNA Synthesis Kit and purified using a miRNeasy Mini Kit;
a Cas9 expression vector is linearized using NotI and transcribed using the mMESSAGE mMACHINE SP6 Kit to produce capped Cas9 RNA.
Preferably, in the aforementioned method, the AAV6 donor vectors contain arms homologous to the β-globin gene of 1.9Kbp on the left side and 0.7Kbp on the right side.
More preferably, in the aforementioned method, the AAV6 donor vectors further contain SV40 polyA as STOP, a reporter gene, and spleen focus forming virus promoters.
More preferably, in the aforementioned method, the reporter gene is linked downstream to the β-globin gene.
More preferably, in the aforementioned method, the reporter gene is EGFP reporter gene or tNGFR reporter gene.
In a fourth aspect, the present application provides a repair system targeting β-globin gene mutation sites constructed by the method according to the third aspect.
In a fifth aspect, the present application provides a universal method to correct types of β-globin gene mutations in β-thalassemia, wherein the universal method uses the repair system according to the fourth aspect.
In summary, the present application provides a gRNA molecule targeting Intron I or Intron II of β-globin gene, a synthetic method thereof, a method for constructing a repair system targeting β-globin gene mutation sites, a repair system, and a universal method to correct types of β-globin gene mutations in β-thalassemia. First, a validated gRNA with a high indel frequency is used as a ribonucleoprotein (RNP) complex to create a DSB in Intron II of the HBB gene. Then, the rAAV6 donor combined with homologous arms is targeted insertion  of 3 exons of the HBB gene in the DSB locus. Additionally, the present application also links a reporter gene downstream to the HBB gene so that the expression of the reporter gene is indicative of successful insertion of the HBB gene into the genome. Using cord blood-derived HSPCs (CB HSPCs) from health donors to test the strategy, inventors find that it can achieve highly efficient targeted insertion and that edited CB HSPCs have normal function compared to noncorrected cells in vitro. Whole-genome sequencing analysis and off-target results indicated that corrected CB HSPCs exhibit a minimal mutational load and no off-target mutagenesis. Moreover, the edited CB HSPCs retain the ability to engraft when transplanted into immunodeficient nonobese diabetic (NOD) -severe combined immunodeficiency (SCID) IL2rg-/- (gamma) mice (NSI mice) ; more importantly, the universal method can correct the β-CD41/42 mutation and improve HBB mRNA expression. In addition, the present application provides an experimental system to screen the small-molecule compounds to improve HDR efficiency in HSPCs based on the co-expression of the reporter gene.
BRIEF DESCRIPTION OF DRAWINGS
The drawings are used to better understand the technical solutions, rather than limiting the present application.
FIG. 1 shows a schematic diagram of gene correction of β-globin gene locus (HBB) using CRISPR/Cas9 and rAAV6; wherein, site-specific DSBs are created by CRISPR/Cas9 (red arrow) . A DSB stimulates homologous recombination (HR) using the rAAV6 homologous donor as a repair template. Light gray boxes: homology arms.
FIG. 2 shows CRISPR/Cas9 mediated targeting of β-globin gene locus (HBB) ; wherein, (A) shows the targeting efficiency of gRNAs targeted to the HBB intron locus in pools of HSC cells was assessed by TIDE; (B) shows sanger sequence of the targeted HBB gene by various gRNAs in HSCs cells; (C) shows the targeting efficiency of mRNA, RNP (Cas9 protein forms ribonucleoprotein with gRNA) or RNP. mgRNA (chemical modified-gRNA ribonucleoprotein ) CRISPR system in HSCs cells, which were assessed by TIDE.
FIG. 3 shows CRISPR/Cas9 and rAAV mediated targeting β-globin gene (HBB)  locus. Specifically, FIG. 3 (A) shows the expression of EGFP reporter gene in HSCs transduced with different MOIs of rAAV6 was analyzed by flow cytometry 10 days after the delivery of RNP. mgRNAiII-2 and RNP. mgRNAiII-3 into HSPCs. FIG. 3 (B) shows flow cytometry results of the EGFP expression in HSCs transduced with 1E+5 MOI rAAV6 after the delivery of RNP. mgRNAII-2 and RNP. mgRNAII-3. FIG. 3 (C) shows a schematic diagram indicating the primer site for the detection of HDR by PCR. FIG. 3 (D) shows agarose gel images of the genotypes of clones targeted at the HBB by rAAV6 after the delivery of RNP. mgRNAiII-2 and RNP. mgRNAiII-3 into HSPCs. FIG. 3 (E) shows the HDR efficiency of EFGP positive HSCs cells transfected with RNP. mgRNAiII-2 and RNP. mgRNAiII-3. FIG. 3 (F) shows the HDR efficiency of all HSCs cells transfected with RNP. mgRNAiII-2 and RNP. mgRNAiII-3.
FIG. 4 shows functional analysis of targeted HSPCs, wherein hematopoietic progenitor CFU assay reveals the forming ability of lineage-restricted progenitors (BFU-E, and CFU-GM) and multipotent progenitors (CFU-GEMM) .
FIG. 5 is a graph of whole exome sequencing of the gene-targeted HSPCs, the graph shows the number of SNVs revealed by whole-exome sequencing in HSPCs cells targeted by RNP. mgRNA; the gray corresponds to background and the red corresponds to mutation sites, the lengths represent genomic density.
DESCRIPTION OF EMBODIMENTS
Exemplary embodiments of the present application are illustrated below with reference to the drawings, various details of the embodiments of the present application are included to facilitate understanding, and they should be considered as merely exemplary. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.
It should be noted that all the methods and processes that are not described in detail in the description use known steps and operations in the art, so they will not be repeated  herein.
The abbreviations used in the embodiments mainly include: HSPCs: Hematopoietic Stem and Progenitor Cells; CRISPR/Cas: Clustered regu larly interspaced short palindromic repeats; RNP: Ribonucleoprotein complex; rAAV6: recombinant adeno-associated virus6; gRNA: small guide RNA; HDR: homology-directed repair; CFU: Colony-Forming Unit; tNGFR: the truncated nerve growth factor receptor; NHEJ: non-homology end joining; DSB: double-strand break. ssODNs: single-strand oligodeoxynucleotides; FACS: Fluorescent-activated cell sorting.
Cell culture
CD34+ HSPCs from cord blood and fetal liver were obtained from the Department of Obstetrics and Gynecology at The Third Affiliated Hospital of Guangzhou Medical University, which was approved by the ethics committee of the hospital. HSPCs were purified within 24 h of scheduled apheresis. Briefly, whole cord blood was mixed with PBS in a proportion of 1: 1 (v/v) , and then the mononuclear fraction was separated by density gradient separation using Ficoll. CD34+ HSPCs were extracted from the mononuclear fraction using a CD34 Microbeads Kit (Miltenyi Biotech, CD34 MicroBead Kit UltraPure, human) according to the manufacturers’ protocol. Cells were stained for CD34 using APC anti-human CD34 (clone 563; BD) to test the purity. All CD34+ HSPCs were cultured in StemSpan SFEMII (StemCell Technologies) supplemented with SCF (100 ng ml  -1) , TPO (100 ng ml  -1) , Flt3 ligand (100 ng ml  -1) , IL-6 (100 ng ml  -1) , Stem Regenin1 (0.75 μM) and UM171 (35 nM) . Cells were cultured at 37 ℃ and 5%CO 2.
AAV vector production
AAV vector plasmids were cloned in the ssAAV-MCS plasmid (PackGene Biotech) , containing inverted terminal repeats (ITRs) from AAV serotype 2 (AAV2) using Gibson Assembly Mastermix (New England Biolabs) . The HBB AAV6 donors contained arms homologous to the beta-globin locus of 1.9Kbp on the left side and 0.7Kbp on the right side (FIG. 1) , the donor also contained SV40 polyA as STOP, a report gene (EGFP or tNGFR) , and spleen focus forming virus promoters. AAV6 vectors were produced as follows: briefly, 1X10 7 293T cells were seeded per 15-cm dish before transfection; each 15-cm dish was transfected with 6 μg of ssAAV-MCS plasmid containing the donor, 7.5 μg of pAAVcap6  containing the AAV6 cap genes and AAV2 rep genes and 7.5 μg of adenovirus helper genes using polyethylenimine (PEI) . After incubating for 72 h, cells were lysed by three freeze-thaw cycles and then incubated with TurboNuclease (Abnova) at 250 U/ml for 45 min. AAV6 particles were purified by iodixanol density gradient centrifugation at 237,000 g for 2 h at 18 ℃. AAV6 vectors were extracted at the 60-40%iodixanol interface and then exchanged in PBS with 5%sorbitol using either a molecular weight cut off (MWCO) Slide-A Lyzer G2 dialysis cassette (Thermo Fisher Scientific) following the manufacturer’s instructions. AAV6 vectors were titered using quantitative PCR to measure the number of vector genomes. The vectors were stored at -80 ℃.
Design and in vitro transcription of gRNAs
The gRNAs targeting Intron I or Intron II of the HBB gene were designed online (http: //crispor. tefor. net/crispor. py) . The gRNAs were cloned into the pUC57-T7 vector (Addgene ID: 51306) ; subsequent sequencing analysis was performed to select the correct gRNA that contained the target site sequence. Templates of the gRNA for in vitro transcription are PCR products obtained from gRNA vectors by using primer pairs, which are T7-F and T7-R, with a high-fidelity enzyme (Takara) ; wherein the sequence of the T7-F is SEQ ID NO: 7, and the sequence of the T7-R is SEQ ID NO: 8. The gRNAs were transcribed using a T7 High Yield RNA Synthesis Kit (NEB) and purified using a miRNeasy Mini Kit (Qiagen) . The Cas9 expression vector was linearized using NotI and transcribed using the mMESSAGE mMACHINE SP6 Kit (Ambion) to produce 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
The HBB gRNAs were produced from in vitro transcription, and the modified gRNA, which had 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 RNP was made by complexing the Cas9 protein with gRNA at a molar ratio of 1: 2.5 (Cas9 protein: gRNA) at room temperature for 10 min before electroporation. CD34+ HSPCs were electroporated using Lonza Nucleofector 2b (program U-014) . The following conditions were used: 5×10 6 cells/ml, 300μg/ml Cas9 protein  complexed with gRNA at 1: 2.5 molar ratio, or 100μg/ml synthetic chemically modified gRNA with 150/μg/ml Cas9 mRNA following electroporation, cells were incubated for 15 min at 37 ℃ after which they were added AAV6 donor vector at various MOI. Then cells were cultured at 37 ℃ and 5%CO 2.
TIDE assays
For measuring the gene targeting efficiency of gRNAs, the PCR products spanning the Cas9-gRNA cleavage site were used for Sanger sequencing, and TIDE software (https: //tide. nki. nl/) was used to quantify the indel frequencies. Primers used for amplifying PCR fragments for TIDE at the beta-globin locus comprise 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’) .
Measuring the targeted integration of fluorescent AAV6 donors and  methylcellulose CFU assay
Rates of targeted integration of GFP donors were measured by flow cytometry 10 days after electroporation. The GFP high populations were sorted into 96-well plates containing MethoCult Optimum (Stem Cell Technologies) by FACS. After 14 days, colonies were counted under an inverted microscope and scored in a blinded fashion based on morphological features of colony forming units-erythroid (CFU-E) , colony forming unit-granulocytes, monocytes (CFU-GM) , and colony forming unit-multipotential cells (CFU-GEMM) .
Genotyping of methylcellulose colonies
Colonies formed in methylcellulose were extracted from FACS sorting of single cells into 96-well plates. Briefly, PBS was added to the 96-well plates, and the colonies were mixed with PBS and transferred to a V-bottomed 96-well plate. Then, the cells were pelleted by centrifugation at 300 g for 5 min at room temperature, and the cells were resuspended in 250 μl of PBS after removing the supernatant. Afterwards, the cells were pelleted by centrifugation at 300 g for 5 min again. Finally, cells were resuspended in 10 μl DNA Extraction Solution and transferred to PCR plates, which were incubated at 65 ℃ for 60 min followed by 95 ℃ for 10 min. PCR was used to detect the integrated alleles, primers are  shown as 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 HSPCs cells using the Cell Genome Extraction kit. SeqCap EZ Exome 64M (Roche NimbleGen) and a TruSeq DNA sample preparation were used to capture the exome and establish the exome sequencing library according to the manufacturers’ instruction manuals. All sequencing was performed on an Illumina NovaSeq with a paired end 2x 150-nucleotide multiplex. Quality control was performed by removing adapter sequences and reads with low complexity or of low quality using Trimmoatic (version 0.39) . Subsequently, BWA (version 0.7.17-r1188) was used to align clean reads to hg38 human genome downloaded from UCSC. Samtools (version 1.9) was used to sort, index bam files and Picard (version 2.20.5) was applied for marking duplicates. GATK (version 4.1.3.0) was used for later steps: 1) base quality score recalibration, 2) variant discovery and 3) variant quality score recalibration. Known sites resource (e.g., SNPs from 1000G Project) was used for Variant Quality Score Recalibration (VQSR) procedure with the parameters (-an QD -an MQ -an MQRankSum -an ReadPosRankSum -an FS -an SOR) , and tranche sensitivity threshold was specified to 90. The variants passed the VQSR procedure were considered to be true variants. Variant annotation and further filtration were carried out using ANNOVAR (version 2019 Oct 24) and whole-genome databases (exac03, avsnp150) . Variants annotated to known sites were eliminated. During the downstream analysis, variants that appeared in all samples were considered as background noise, and were eliminated in further analysis. Genomic tracks of mutations were drawn using Circlize (version 0.4. ) was used for other customized visualizations.
Development of a universal approach targeting the HBB gene
In order to develop a universal method to correct most HBB mutations, inventors tested the scheme shown in FIG. 1. Briefly, inventors designed a method to achieve HDR in Intron I or Intron II of the HBB locus. Site-specific DSB was created by the ribonucleoprotein (RNP) complex consisting of gRNA and Cas9 protein, and HDR was achieved using an rAAV6 homologous donor as a repair template. To facilitate enriching and tracing HDR,  inventors further added an EGFP or tNGFR reporter gene downstream of the HBB gene.
Optimizing the delivery of Cas9/gRNA RNP into hematopoietic  stem/progenitor cells
In the present application, inventors selected Six gRNAs with high scores targeting the two introns of HBB using the web-based search tool. gRNA II-2 and gRNA II-3 showed the highest on-target efficiency in pools of HSPCs electroporated with a plasmid (FIG. 2A, B) . Previous studies have clarified that the Cas9/gRNA system delivered as an RNP complex by electroporation is the most effective method for creating DSBs and stimulating HR in HSPCs [14, 19] . Moreover, it has previously been shown that 2’-O-methyl-3’-phosphorothioate (MS) modifications to the three terminal nucleotides at the 5’ and 3’ ends of the gRNA significantly increase the ability of the Cas9/gRNA system to induce DSBs in HSPCs [16, 20] . Therefore, the present application determined whether modified gRNA and RNP was more effective than mRNAs. By introducing the Cas9-mRNA/gRNA, Cas9/gRNA RNP and Cas9/mgRNA RNP into HSPCs by electroporation and harvesting cells four days later, the present application found a significant indel ratio increase generated at the HBB intro locus when the Cas9/gRNA system was delivered as an RNP with modified guides versus unmodified guides (FIG. 2C) . Above all, the targeting efficiency the HBB intro locus can reach to 80%.
Optimizing rAAV6 donor transduction into HSPCs to achieve consistently  high levels of HDR
The inventors next optimized rAAV6 donor transduction by titering the MOI or vector genomes/cell (vgs/cell) that stimulated the highest rates of HDR in HSPCs with an appropriate cell survival rate. Following the delivery of Cas9/gRNA RNP to cells by electroporation, CB-HSPCs were transduced with the rAAV6 donor at MOIs ranging from 1×10 3-1×10 6 MOI. The inventors found that the rates of EGFP positive reached to 20%at an MOI of 1×10 5 (FIG. 3A, B) , and the rates of HDR were not increased with increasing MOI. An MOI of 1×10 5 for the following experiments was chosen.
Identify the genotype of edited clones at the HBB locus
In order to identify the genotype of HBB-targeted HSPCs, the inventors performed single-cell methylcellulose cloning of populations. After 10 days HSPCs transduced with RNP. mgRNA and rAAV6, EGFP positive cells were sorted and seeded in methylcellulose  medium for 2 weeks. For detecting the targeted integration in HBB locus, primers were designed outside of the homologues arms (FIG. 3C) . In the EGFP positive cells, about 60%-85%cells had targeted integration (FIG. 3E, D) . Overall, 10%-20%of the trans ducted HSPCs cells were targeted integration (FIG. 3F) .
Functional analysis of CB HSPCs after delivery with RNP and rAAV6 donor
In order to observe whether the HBB-targeted strategy can influence the function of HSPCs, the inventors performed a hematopoietic progenitor CFU assay to show the forming ability of lineage-restricted progenitors (BFU-E, and CFU-GM) and multipotent progenitors (CFU-GEMM) . The data demonstrated that there was no significant difference between the untargeted and targeted EGFP positive cells (FIG. 4A) . Above all, our data suggested that the HBB-targeted strategy did not influence the function of HSPCs.
Whole exome sequencing analysis of gene-targeted HSPCs
CRISPR/Cas9 has highly improved gene targeting efficiency, but it has a potential risk of causing off-target mutagenesis. Due to the importance of exon regions, which affect most cell biological functions, the inventors performed the whole exome sequencing. Genomic DNA from sorted EGFP positive cells were used for NGS library construct. Mutations detected in all samples were considered as background somatic mutations. There was no significant mutations sited were detected in both RNP. mgRNAII-2 and RNP. mgRNAII-3 (FIG. 5) .
As described above, the present application has established an efficient and universal repair system targeting β-globin gene mutation sites using the Cas9 RNP in combination with rAAV6 homologous donor vectors. Furthermore, the method to correct types of β-globin gene mutations provided by the present application enables correction of not only HBB mutations but also some upstream and downstream mutations in the HBB gene.
Additionally, in the process of optimizing the targeting efficiency of our RNP and rAAV6 donor, the present application finds that the modified synthetic gRNA is significantly higher than IVT gRNA. The targeting efficiency were archived to 80%, which is beneficial because the modified synthetic gRNA is not degraded by the cellular immune response, and chemical modification provides greater stability and protection from exonucleases. This is a finding that our group and others have also demonstrated in the prior art [24, 25] . A higher  indel ratio is the basis of high HDR efficiency, and it is nearly 16%HDR efficiency in the present application. The present application finds that the targeted HSPCs hold similar differential ability in vitro compare with untargeted cells. And there is no off-target effect that is related to the repair system by whole exome sequencing.
In conclusion, the present application provides a universal strategy to correct most types of HBB gene mutations in HSPCs. The targeted HSPCs have normal function in vitro and no significant off-target effect.
References of the present application are listed as follows: [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 in β-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 with β-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, 
Figure PCTCN2021070839-appb-000002
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 of β-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.
The above specific embodiments do not limit the protection scope of the present application. Those skilled in the art should understand that various modifications, combinations, sub-combinations and substitutions can be made based on the actual requirements and other factors. Any modification, equivalent alternative, or improvement within the spirit and principle of the present application shall be regarded as falling within the protection scope of the present application.

Claims (16)

  1. A gRNA molecule targeting Intron I or Intron II of β-globin gene, wherein the gRNA molecule is selected from the group consisting of 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 as 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 as SEQ ID NO: 3, the sequence of gRNA II-1 is shown as SEQ ID NO: 4, the sequence of gRNA II-2 is shown as SEQ ID NO: 5, and the sequence of gRNA II-3 is shown as SEQ ID NO: 6.
  2. The gRNA molecule according to claim 1, wherein the gRNA molecule is gRNA II-2 or gRNA II-3 targeting Intron II of β-globin gene.
  3. A synthetic method of the gRNA molecule according to claim 1 or 2, wherein templates of the gRNA molecule for in vitro transcription are PCR products obtained from gRNA vectors by using primer pairs, which are T7-F and T7-R, with a high-fidelity enzyme; wherein the sequence of the T7-F is SEQ ID NO: 7, and the sequence of the T7-R is SEQ ID NO: 8.
  4. A method for constructing a repair system targeting β-globin gene mutation sites, comprising steps of:
    S1: synthesizing Cas9/gRNA RNP;
    S2: performing electroporation of cells;
    S3: incubating electroporated cells after they are added AAV6 donor vectors at various MOI (Multiplicity of Infection) ;
    S4: culturing incubated cells;
    wherein, the gRNA is selected from the group consisting of 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 as 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 as SEQ ID NO: 3, the sequence of gRNA II-1 is shown as SEQ ID NO: 4, the sequence of gRNA II-2 is shown as SEQ ID NO: 5, and the sequence of gRNA II-3 is shown as SEQ ID NO: 6.
  5. The method according to claim 4, wherein the Cas9/gRNA RNP is made by complexing Cas9 protein with the gRNA at a molar ratio of 1: 2.5 at room temperature.
  6. The method according to claim 4, wherein the cells in S2 is CD34+ HSPCs (Hematopoietic Stem and Progenitor Cells) .
  7. The method according to claim 4, wherein the electroporated cells are incubated at 37 ℃ in S3.
  8. The method according to claim 4, wherein the MOI ranges from 1×10 3 to 1×10 6, preferably 1×10 5, in S3.
  9. The method according to claim 4, wherein the incubated cells are cultured at 37 ℃ and 5%CO 2 in S4.
  10. The method according to claim 4, wherein before S1, in vitro transcription of the gRNA comprises:
    the gRNAs are cloned into the pUC57-T7 vector (Addgene ID: 51306) ;
    subsequent sequencing analysis is performed to select the correct gRNA that contained target site sequence; wherein, templates of the gRNA for in vitro transcription are PCR products obtained from gRNA vectors by using primer pairs, which are T7-F and T7-R, with a high-fidelity enzyme; wherein the sequence of the T7-F is SEQ ID NO: 7, and the sequence of the T7-R is SEQ ID NO: 8;
    and then, the gRNA is transcribed using a T7 High Yield RNA Synthesis Kit and purified using a miRNeasy Mini Kit;
    a Cas9 expression vector is linearized using NotI and transcribed using the mMESSAGE mMACHINE SP6 Kit to produce capped Cas9 RNA.
  11. The method according to claim 4, wherein the AAV6 donor vectors contain arms homologous to the β-globin gene of 1.9Kbp on the left side and 0.7Kbp on the right side.
  12. The method according to claim 11, wherein the AAV6 donor vectors further contain SV40 polyA as STOP, a reporter gene, and spleen focus forming virus promoters.
  13. The method according to claim 12, wherein the reporter gene is linked downstream to the β-globin gene.
  14. The method according to claim 12, wherein the reporter gene is EGFP reporter gene or tNGFR reporter gene.
  15. A repair system targeting β-globin gene mutation sites constructed by the method according to any one of claims 4-14.
  16. A universal method to correct types of β-globin gene mutations in β-thalassemia, wherein the universal method uses the repair system according to claim 15.
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