A method, product and application for gene editing of single or multiple genes in cells

Abstract

The present invention discloses a method, product and application for gene editing of multiple genes in cells. The method includes the step of introducing an adenine base editor and at least one sgRNA into a cell; the at least one sgRNA includes any one, any two or all three of sgRNA1, sgRNA2 and sgRNA3, and the characteristics of the sgRNA1-3 are as described in the present invention. The present invention can utilize gene editing technology in combination with autologous hematopoietic stem cell transplantation to treat transfusion-dependent β-thalassemia (Transfusion-dependent β-thalassemia, TDT) and sickle cell disease (sickle cell disease, SCD). The technology used in the present invention has extremely high gene editing efficiency in hematopoietic stem / progenitor cells (HSPC), and the HSPCs edited by the gene can be differentiated into blood cells of each lineage after transplantation in vivo, rebuilding the hematopoietic system, while the expression of fetal hemoglobin in the red blood cells differentiated from the HSPCs edited by the gene is greatly improved, and the survival of the red blood cells in the body and the performance of normal physiological functions are also promoted.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a method, product and application for gene editing of single or multiple target genes in cells. Background Art

[0002] Causes, hazards, and treatment status of thalassemia and sickle cell anemia

[0003] Hemoglobin is composed of two pairs of protein chains. Under normal circumstances, adults have one pair of α chains and one pair of β chains. Severe transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) are two serious diseases caused by mutations in the HBB gene encoding β-globin, resulting in a lack of hemoglobin β chain quantity and functional loss. IVS-II-654C>T genotype HBB is a more common type of thalassemia in my country. The pathogenesis is a C>T mutation at the 654th base in the second intron of the HBB gene, which produces an abnormal splicing site, resulting in an additional 73nt exon in the mRNA encoding β-globin and premature termination of translation, that is, it cannot be translated into a normal β chain, resulting in an imbalance in the quantity of α and β chains of adult hemoglobin. The second most common thalassemia gene mutation in my country is CD41-42. Its pathogenesis is a four-base deletion, TTCT, in the HBB gene encoding amino acids 41 and 42. This mutation results in a reading frame mutation, premature stop codons, and virtually no normal β-chain synthesis, leading to an imbalance in the number of α and β chains in adult hemoglobin. SCD is a homozygous abnormal hemoglobin (hemoglobin S (HbS)) caused by an A>T genotype mutation in the HBB gene, which results in a substitution of valine for glutamic acid at position 6 of the encoded β-globin chain. This results in a hemoglobin tetramer (α2 / βS2, or HbS) that is virtually insoluble when deoxygenated. The hallmark features of SCD are vaso-occlusive phenomena and hemolytic anemia. The aggregation of deoxygenated HbS is essential for vaso-occlusive phenomena. Vaso-occlusive events can lead to recurrent painful episodes (formerly known as sickle cell crises) and a variety of serious organ system complications that can cause lifelong disability and / or premature death. These aggregates appear as long, rope-like fibers that often connect to other fibers, twisting the red blood cells into their characteristic crescent or sickle shapes and significantly reducing their deformability. The red blood cells of SCD patients only carry half as much oxygen as normal red blood cells, and the red blood cells of patients with beta-thalassemia cannot carry oxygen as effectively. Both SCD and thalassemia major are serious inherited blood disorders. People with these conditions require lifelong blood transfusions, injections, or medication.

[0004] TDT and SCD can be cured through allogeneic stem cell or bone marrow transplantation, but this is limited by donor availability and carries a high risk: the recipient may reject the donated bone marrow or cells, leading to severe side effects or even death. Consequently, allogeneic stem cell or bone marrow transplantation for TDT and SCD is not widely used clinically.

[0005] The principle of gene editing HBG1 / 2 genes for disease treatment

[0006] The HBG1 / 2 genes encode γ-globin, a globin protein expressed during the fetus that has similar functions to β-globin. The HBG1 / 2 genes encoding this protein are intact in patients with the aforementioned anemia, but their expression is silenced in adulthood. Clinical studies have shown that the severity of β-thalassemia is closely correlated with the level of fetal hemoglobin (HbF), which consists of a pair of γ-globin and a pair of α-globin chains. In some patients, higher levels of HbF are associated with milder complications and lower mortality. Therefore, increasing HbF levels (HbF: α2γ2) is an important approach to treating or alleviating this type of anemia. Increasing fetal globin levels can alleviate the globin chain imbalance in hemoglobin in patients with β-thalassemia and potentially inhibit sickling in patients with sickle cell anemia. Furthermore, in most patients with hemoglobinopathies, the upstream γ-globin genes HBG1 / 2 are intact and fully functional. Reactivating these genes can maintain functional hemoglobin HbF synthesis in adulthood, thereby improving disease severity. Therefore, reactivating the expression of the γ-globin gene in patients to compensate for the defective β-globin is a therapeutic strategy that has emerged in recent years.

[0007] Previous studies have found that in patients with β-thalassemia, mutations or large deletions in specific regions of the HBG1 / 2 gene promoter cluster lead to a certain amount of γ-globin gene expression, compensating for the missing β-globin and alleviating the patient's symptoms. These mutations are known as hereditary persistence of fetal hemoglobin (HPFH), and the corresponding mutations are known as HPFH mutations. Currently, identified HPFH mutations that are associated with adult HbF expression and increase HbF expression to 40% of total Hb (hemoglobin) expression, as well as single-nucleotide polymorphisms (SNPs), are located in the -200, -175, -158, and -115 bp regions (e.g., the -102 to -114 bp deletion) of the HBG1 gene, as well as in the transcription start region of the HBG2 gene. HPFH mutations and SNPs can form new DNA motifs in the HbF gene that are recognized by transcriptional activators or directly disrupt the binding region of transcriptional repressors in the HbF gene, thereby enhancing HbF expression. Notably, HPFH mutations near the -200 bp and -115 bp regions disrupt or weaken the binding of the transcriptional repressors LRF and BCL11A, respectively, thereby abolishing γ-globin silencing. Furthermore, SNPs near the -158 bp position in the promoter region of the γ-globin-encoding genes HBG1 / 2 are closely associated with enhanced γ-globin expression. Therefore, editing the promoter region of HBG1 / 2 to mimic natural HPFH mutations and SNPs and activate γ-globin expression is a potential therapeutic strategy for TDT and SCD.

[0008] Therefore, to mimic these natural HPFH and SNPs, we used ABE base editors to target the natural mutation region in the promoter region of the HBG1 / 2 gene that has been found to be beneficial for disease treatment in reported studies, and efficiently introduced A>G or T>C base mutations to simulate the effect of increased HbF levels brought about by natural HPFH mutations, thereby achieving the purpose of disease treatment.

[0009] BCL11A is a silencer that suppresses γ-globin gene expression in adult erythrocytes

[0010] Previous studies have confirmed that BCL11A is a silencer that suppresses γ-globin gene expression in adult erythrocytes. In 2013, Daniel E. Bauer et al., through genome-wide association studies (GWASs), found that common mutations in the noncoding regions of the BCL11A protein-coding gene, characterized by chromatin signatures of erythroid enhancers, were associated with fetal hemoglobin (HbF) expression levels. The SNPs most strongly associated with HbF levels were located within three DNase I hypersensitive sites (DHSs) within the BCL11A enhancer: +62 kb, +58 kb, and +55 kb (62 kb, 58 kb, and 55 kb downstream of the transcription start site (TSS)).

[0011] In 2015, Daniel E. Bauer and other researchers conducted Cas9-mediated saturation knockout experiments in the 62kb, 58kb, and 55kb regions downstream of the transcription start site (TSS) in the BCL11A enhancer region. They found that the +62kb, +58kb, and +55kb regions are important functional regions. For example, Cas9 cutting the +58kb region can almost knock out the BCL11A gene, and the proportion of cells expressing fetal hemoglobin HbF also increases accordingly. In 2019 and 2020, Wu Yuxuan and other researchers used Cas9 or CBE to edit the +58kb region of the BCL11A enhancer in HSPCs, respectively, and confirmed that cutting or mutation of the GATA / half E-box motif at the +58kb site will produce red blood cells with high expression of HbF without affecting the differentiation and maturation of lymphocytes.

[0012] Two articles were published simultaneously in the New England Journal on December 5, 2020. One of them used lentivirus to infect CD34+ cells obtained from sickle cell disease donors in vitro, and then infused the infected autologous CD34+ cells back into the sickle cell disease patients. The shRNA expressed by the lentiviral vector specifically targeted and knocked down the mRNA of the BCL11A gene in CD34+ cells, thereby reactivating the HBG1 / 2 gene and maintaining functional hemoglobin HbF synthesis in the adult stage. The clinical manifestations of sickle cell disease in the six treated patients were alleviated or disappeared during the follow-up period (median time of 18 months). Among them, five patients no longer needed blood transfusions, and one patient began to receive blood transfusion therapy after 18 months of treatment. Secondly, the research team electroporated CD34+ hematopoietic stem / progenitor cells obtained from patient donors and used CRISPR-Cas9 to target the BCL11A erythroid-specific enhancer for editing. Subsequently, two patients (one with β-thalassemia and one with sickle cell anemia) received a transfusion of autologous CD34+ cells that had been edited with CRISPR-Cas9 to modify the BCL11A enhancer. More than a year later, both patients had high levels of edited alleles in their bone marrow and blood, with 99% of circulating cells expressing fetal hemoglobin (HbF) and no longer dependent on blood transfusions. The SCD patient also experienced resolution of vaso-occlusive episodes.

[0013] The two studies reported in the New England Journal of Medicine achieved the same goal through different approaches. They used different technical means to reduce the expression of BCL11A protein, thereby reactivating the expression of γ-globin, and ultimately increasing the level of fetal hemoglobin (HbF) and eliminating dependence on blood transfusions.

[0014] The principle of gene editing TFR2 gene for disease treatment

[0015] The transferrin receptor (TFR) in plasma is a key protein in cellular iron uptake from transferrin. Two TFRs are currently known: TFR1 and TFR2. The latter molecule was recently discovered, and its function remains unclear. Both receptors bind to transferrin, which carries two iron atoms, in a pH-dependent manner, transporting iron into cells. However, the affinity of TFR2 is 25-fold lower than that of TFR1. TFR2 is a protein that regulates iron transport and iron homeostasis. TFR2 shares similarities with TFR1 in gene structure, protein structure, binding to transferrin (Tf), and iron uptake, but differs significantly from TFR1 in terms of tissue distribution and regulation of mRNA expression. TFR2 mRNA is expressed at high levels in hepatocytes and erythroid progenitor cells. TFR2 gene transcripts have at least two alternatively spliced forms: α and β. TFR2-α is similar to TFR1. TFR2-α is expressed on the cell surface and specifically binds to Tf, promoting its uptake into cells. The TFR2-β protein lacks intracellular and transmembrane domains and may be an intracellular protein. Its function is currently unclear. Therefore, the TFR2 mentioned below in this invention specifically refers to TFR2-α. The TFR2-α protein is a membrane protein composed of 801 amino acids with a molecular weight of approximately 105kDa. The TFR2 protein plays an important role in iron balance in erythroid precursor cells, and reported studies have found that knocking out the TFR2 gene is beneficial to the relief of anemia symptoms in intermediate β-thalassemia model mice; therefore, editing the TFR2 gene will become one of the new potential strategies for the treatment of β-thalassemia.

[0016] ABE base editing technology principles and advantages

[0017] Base editor technology, developed based on the CRISPR / Cas system, is a novel targeted gene modification technique that can introduce single-base mutations at the target site without introducing double-strand breaks (DSBs). Base editors primarily consist of two components: the Cas protein and the DNA-modifying enzyme. David R. Liu's group developed the cytosine base editor (CBE) and adenine base editor (ABE) in 2016 and 2017, respectively. These two base editing systems utilize cytosine deaminases (CBEs) or artificially evolved adenine deaminases (ABEs) to perform precise base editing at the target site, ultimately achieving: cytosine base editors, which replace C·G with T·A; and adenine base editors, which replace A·T with C·G. The seventh-generation adenine base editor, ABE7.10, achieved the highest efficiency during initial ABE development, efficiently converting A·T to C·G, with efficiency reaching up to 50% at certain sites in human 293T cells.

[0018] To further enhance the compatibility and activity of ABEs with diverse Cas homologs, researchers from David R. Liu's group reported in March 2020 the development of an eighth-generation ABE mutant, ABE8e, using a novel phage-assisted evolution system. Compared to ABE7.10, ABE8e's deaminase harbors eight additional mutations, resulting in a 590-fold increase in deamination activity. The researchers significantly enhanced the editing efficiency of ABE8e by pairing the deaminase with various Cas homologs, including SpCas9.

[0019] Advantages of ABE editing HBG1, BCL11A, and TFR2 genes for disease treatment

[0020] Currently, patients with thalassemia intermedia and major require long-term blood transfusions and iron chelation therapy to survive. The only cure is allogeneic hematopoietic stem cell transplantation, but major obstacles to implementation include the scarcity of blood resources in my country, difficulties in matching allogeneic hematopoietic stem cells, and transplant-related complications. Gene therapy using lentiviral vectors and Cas9 protein electroporation for thalassemia intermedia and major shows great potential. However, the semi-random integration of lentiviral vectors carries the risk of carcinogenesis, while the cleavage mechanism of Cas9 protein can introduce unintended random insertions, deletions, and mutations, compromising the purity of the edited product and posing risks. Furthermore, the expression elements in the lentiviral vectors can become silenced during the long-term homing and self-renewal of hematopoietic stem cells, resulting in decreased efficacy and the potential failure to achieve a lifelong cure. Furthermore, the high-concentration, high-quality lentiviruses required for clinical use place extremely high demands on the equipment and technology, making them difficult to reduce in cost. Therefore, parallel, safer, and more cost-effective clinical approaches are highly desirable.

[0021] The ideal gene therapy method is to repair the thalassemia mutation on the HBB gene in the patient's hematopoietic stem cell DNA or activate the HBG1 / 2 gene, and permanently produce wild-type adult γ-globin or β-globin under the action of endogenous transcription control factors, so that hematopoietic stem cells can differentiate into normal functional red blood cells.

[0022] The strategy of using the ABE editing system to treat thalassemia and sickle cell anemia has some advantages over the two research strategies reported in the New England Journal of Medicine on December 5, 2020: Compared with Cas9, ABE has a significantly lower off-target rate and is therefore safer, and does not cause double-strand breaks in DNA, and thus does not produce non-ideal insertions and deletions (indels) and P53 reactions, etc.; and one of the preferred delivery methods included in the present invention - the RNP electroporation method, also has some advantages - Lentiviral lentivirus cannot control the gene insertion point, and it can be simply considered that the insertion point is "randomly" selected. Since the insertion point is "random", the number of exogenous genes inserted into a single cell is also uncertain. Therefore, the integration of exogenous genes into certain sites of the genome may lead to the occurrence of cancer. Compared with the lentiviral infection system, the RNP delivery method of ABE is safer. The present invention targets abnormal mutation regions of pathogenic sites or mutation regions that are beneficial to disease treatment. It only requires the use of a single-base editor to introduce therapeutically beneficial mutations or repair the original pathogenic mutations, and the strategy appears to be more feasible. In clinical practice, cure can be achieved by simply transplanting gene-edited autologous hematopoietic stem cells back into the body. Summary of the Invention

[0023] The present invention aims to provide a method, product, and application for gene editing of single or multiple genes in cells. The genes include HBG1 / 2, BCL11A, and TFR2, and the gene editing utilizes ABE editing. The present invention can be used to treat and / or prevent β-thalassemia, sickle cell anemia, and / or dyserythropoiesis.

[0024] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0025] The present invention provides a method for gene editing at least one of the HBG1 / 2 gene, the BCL11A gene, and the TFR2 gene in a cell, comprising the steps of introducing an adenine base editor and at least one sgRNA into the cell;

[0026] The at least one sgRNA is any one, any two or all three of sgRNA1, sgRNA2 and sgRNA3,

[0027] Any one of: sgRNA1, sgRNA2, or sgRNA3,

[0028] Any two of them are: sgRNA1 and sgRNA2, sgRNA1 and sgRNA3, or sgRNA2 and sgRNA3,

[0029] The three types are: sgRNA1, sgRNA2 and sgRNA3,

[0030] The sgRNA1 guides the adenine base editor to perform A>G and / or T>C editing on bases within 20 kb of the HBG1 gene promoter region of γ-globin and / or bases within 20 kb of the HBG2 gene promoter region to activate or enhance the expression of fetal hemoglobin HbF (HbF: α2γ2);

[0031] The sgRNA2 guides the adenine base editor to perform A>G and / or T>C editing on the bases within the enhancer region of the γ-globin transcription repressor BCL11A gene to activate or enhance the expression of fetal hemoglobin HbF (HbF: α2γ2).

[0032] The sgRNA3 guides the adenine base editor to perform A>G and / or T>C editing on the bases within the transferrin receptor TFR2 gene to knock out or knock down the expression of transferrin receptor TFR2.

[0033] In the above method, preferably, the adenine base editor comprises an amino acid sequence as shown in SEQ ID No: 25 (ABE8e), or a sequence having 80%, 85%, 90%, 95%, 97%, 98%, 99% or more homology with the sequence shown in SEQ ID No: 25 and has at least the same or similar function as the adenine base editor as shown in SEQ ID No: 25.

[0034] Preferably, the amino acid sequence of the adenine base editor is shown in SEQ ID No: 25.

[0035] The inventors applied ABE8e to human CD34+ hematopoietic stem / progenitor cells and found that ABE8e can effectively introduce an A>G mutation in the enhancer region of the BCL11A gene in human cells, while the editing efficiency of ABE7.10 at the same site is very poor.

[0036] In other embodiments, the adenine base editor includes a base editor protein with higher efficiency, lower off-target rate, and better accuracy with more amino acid mutations obtained by further protein evolution based on the ABE8e protein expression sequence;

[0037] In other embodiments, other deaminases and mutants thereof may be used to replace the deaminase TadA mutant portion in the ABE8e protein;

[0038] In other embodiments, other Cas enzymes and mutants can be used to replace the nCas9 portion of the ABE8e protein;

[0039] In other embodiments, some mutations can be introduced into the Cas enzyme part including Cas9 to weaken or remove the PAM restriction of ABE8e and thus expand the editing range.

[0040] In the above method, preferably, the target sequence of the sgRNA1 includes any sequence within the range of -90 bp to -220 bp of the HBG1 gene promoter region and / or any sequence within the range of -90 bp to -220 bp of the HBG2 gene promoter region;

[0041] Preferably, the target sequence of the sgRNA1 includes at least one of the sequences shown in SEQ ID No: 1-8;

[0042] More preferably, the targeting sequence of the sgRNA1 includes the sequence shown in SEQ ID No: 1.

[0043] In the above method, the targeting sequence of the sgRNA2 includes at least one of the DNase I hypersensitive sites (DHS) +62kb, +58kb and +55kb ("+" represents downstream of the BCL11A gene transcription start site (TSS)) in the enhancer region of the BCL11A gene and any sequence within the nearby 20kb region.

[0044] Preferably, at least one of the DNase I hypersensitive sites +62kb, +58kb and +55kb in the enhancer region of the BCL11A gene and a half E-box sequence and / or a GATA motif within the 20kb region nearby and any sequence in the nearby region; More preferably, the target sequence targeted by the sgRNA2 includes the DNase I hypersensitive site +58kb and / or +55kb half E-box sequence and / or a GATA motif within the enhancer region of the BCL11A gene and any sequence in the nearby region;

[0045] Further more preferably, the target sequence of the sgRNA2 includes at least one of the sequences shown in SEQ ID No: 9-13 in the +58kb region and / or at least one of the sequences shown in SEQ ID No: 14-15 in the +55kb region,

[0046] More preferably, the target sequence targeted by the sgRNA2 includes the sequence shown in SEQ ID No: 11 and / or SEQ ID No: 14; or the target sequence targeted by the sgRNA2 includes the sequence shown in SEQ ID No: 12 and / or SEQ ID No: 14.

[0047] In a best embodiment of the present invention, the target sequence of the sgRNA2 includes the sequence shown in SEQ ID No:14.

[0048] In the above method, preferably, the target sequence of the sgRNA3 includes the translation start codon (ATG) of the TFR2 gene and the following 5 non-frameshifted ATGs and any sequence in the vicinity thereof; preferably, the target sequence of the sgRNA3 includes at least one of the sequences shown in SEQ ID No: 16-20; more preferably, the target sequence of the sgRNA3 includes the sequence shown in SEQ ID No: 16, or,

[0049] The target sequence of the sgRNA3 includes any sequence within the intron splice donor site region of the TFR2 gene. Preferably, the target sequence of the sgRNA3 includes any sequence within the intron splice acceptor site region before exon 4, 7, 8, or 15 of the TFR2 gene and its vicinity. More preferably, the target sequence of the sgRNA3 includes at least one of the sequences shown in SEQ ID Nos: 21 to 24. More preferably, the target sequence of the sgRNA3 is the sequence shown in SEQ ID No: 23.

[0050] In another embodiment, double-stranded DNA breaks mediated by sgRNA including Cas9 or other Cas enzymes can be used to disrupt the TFR2 encoding gene to achieve gene knockout or knockdown expression.

[0051] In another embodiment, a cytosine base editor CBE can be used to achieve C>T (or G>A in the complementary chain) mediated by sgRNA, thereby introducing the stop codon UAA or UAG or UGA in advance before the normal stop codon of TFR2, thereby terminating the translation of TFR2 protein in advance and producing a non-functional protein.

[0052] In another embodiment, a cytosine base editor CBE can be used to achieve C>T (or G>A in the complementary chain) mediated by sgRNA, and the "CT" of the reverse complementary chain of the intron splicing acceptor region - "AG" (acceptor) can be edited to edit the "C" in "CT" to "T", thereby causing splicing problems and abnormal protein expression, thereby destroying protein function.

[0053] In the above method, the cells preferably include hematopoietic stem / progenitor cells or erythroid progenitor cells; more preferably, the hematopoietic stem / progenitor cells include CD34+ hematopoietic stem / progenitor cells.

[0054] In the above method, the cells are preferably derived from mammals, more preferably from humans or mice.

[0055] Preferably, the sgRNA comprises chemical modifications of bases.

[0056] In a preferred embodiment, the sgRNA comprises chemical modifications of any one or several bases at positions 1 to n at the 5' terminus, and / or chemical modifications of any one or several bases at positions 1 to n at the 3' terminus; wherein n is selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the sgRNA comprises chemical modifications of one, two, three, four, or five bases at the 5' terminus, and / or chemical modifications of one, two, three, four, or five bases at the 3' terminus. For example, the 1st base, the 2nd base, the 3rd base, the 4th base, the 5th base, or the 1-2nd base, the 1-3rd base, the 1-4th base, and the 1-5th base at the 5' end of the sgRNA is chemically modified; and / or the 1st base, the 2nd base, the 3rd base, the 4th base, the 5th base, or the 1-2nd base, the 1-3rd base, the 1-4th base, and the 1-5th base at the 3' end of the sgRNA is chemically modified; in a preferred embodiment, the chemical modification is one or any combination of methylation, fluorination, and thiolation.

[0057] Preferably, the introducing of the adenine base editor and at least one sgRNA into the cell comprises an electroporation method, specifically an electroporation method for introducing a complex comprising the adenine base editor and at least one sgRNA into the cell.

[0058] Furthermore, the molar ratio of the adenine base editor to the at least one sgRNA is 1:(1-3), preferably 1:2 or 1:3.

[0059] Furthermore, the adenine base editor and the at least one sgRNA form a complex by incubation; preferably, the incubation temperature is 20-50°C, more preferably 25-37°C; preferably, the incubation time is 2-30 minutes, preferably 5-20 minutes, more preferably 10 minutes.

[0060] Furthermore, the ratio of the amount of the complex comprising the adenine base editor and the at least one sgRNA to the cells is 20-100 μg complex: (1×10 2 -1×10 6 cells, preferably 30 μg of complex: (1×10 3 -1×10 5 cells;

[0061] The electroporated cells were then cultured in a CD34+ EDM-1 differentiation system for 7 days. After the fourth day of in vitro differentiation, genomic DNA was extracted from the cells obtained above for genotyping and mutation efficiency determination. After the mutation efficiency was determined, differentiation was continued to the EDM-2 stage for 4 days and the EMD-3 stage for 7 days. On days 3-4 of differentiation, appropriate cells were harvested, total protein was extracted, and TFR2-α and TFR2-β were analyzed by Western blot. On day 18 of differentiation, RNA was extracted from a portion of the erythrocytes and reverse-transcribed into cDNA. The mRNA levels of HBG1 / 2 genes were analyzed by qPCR. The remaining erythrocytes were lysed with ice water, and the supernatant was centrifuged and analyzed for γ-globin expression from the HBG1 / 2 genes by HPLC.

[0062] In other embodiments, the method of introducing the adenine base editor and at least one sgRNA into cells can also be vector transformation, transfection, heat shock, electroporation, transduction, gene gun, and microinjection.

[0063] In another aspect, the present invention provides a recombinant cell prepared by any of the above methods.

[0064] Preferably, the recombinant cells are hematopoietic stem / progenitor cells or erythroid progenitor cells; more preferably, the hematopoietic stem / progenitor cells are CD34+ hematopoietic stem / progenitor cells.

[0065] Preferably, the recombinant cell is derived from a mammal, more preferably, from a human or a mouse.

[0066] On the other hand, the present invention provides a single-base gene editing system, which comprises the adenine base editor defined above and at least one sgRNA defined above.

[0067] In another aspect, the present invention provides an sgRNA composition comprising at least one sgRNA as defined above.

[0068] Preferably, the sgRNA composition comprises an sgRNA targeting any one or more sequences selected from SEQ ID NOs: 1, 4 to 6, 8, 11, 12, 14, 16 and 23.

[0069] More preferably:

[0070] The sgRNA composition comprises an sgRNA targeting any one or more sequences selected from SEQ ID NO: 1, 11, 12, 14, and 16;

[0071] The sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12 and an sgRNA targeting the sequence shown in SEQ ID NO: 1;

[0072] The sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12 and an sgRNA targeting the sequence shown in SEQ ID NO: 4;

[0073] Alternatively, the sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12 and an sgRNA targeting the sequence shown in SEQ ID NO: 5;

[0074] Alternatively, the sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12 and an sgRNA targeting the sequence shown in SEQ ID NO: 6;

[0075] Alternatively, the sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12 and an sgRNA targeting the sequence shown in SEQ ID NO: 8;

[0076] Alternatively, the sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, an sgRNA targeting the sequence shown in SEQ ID NO: 1, and an sgRNA targeting the sequence shown in SEQ ID NO: 16;

[0077] Alternatively, the sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 14;

[0078] Alternatively, the sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 14 and an sgRNA targeting the sequence shown in SEQ ID NO: 1;

[0079] Alternatively, the sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 14 and an sgRNA targeting the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12;

[0080] Alternatively, the sgRNA composition comprises an sgRNA targeting the sequence shown in SEQ ID NO: 14, an sgRNA targeting the sequence shown in SEQ ID NO: 1, and an sgRNA targeting the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12.

[0081] On the other hand, the present invention protects the use of any of the above methods, the recombinant cells, the single-base gene editing system, and / or the sgRNA composition in the preparation of gene editing products, disease treatment and / or prevention products, or animal models.

[0082] Preferably, the cells targeted by the gene editing product include hematopoietic stem / progenitor cells or erythroid progenitor cells; more preferably, the hematopoietic stem / progenitor cells include CD34 + hematopoietic stem / progenitor cells.

[0083] Preferably, the cells are derived from mammals, more preferably from humans or mice.

[0084] Preferably, the disease comprises beta-thalassemia and / or sickle cell anemia and / or dyserythropoiesis.

[0085] The "nearby 20kb region" or "nearby region" mentioned above is a region with a length of 20kb, 19kb, 18kb, 17kb, 16kb, 15kb, 14kb, 13kb, 12kb, 11kb, 10kb, 9kb, 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1k, or 0.5kb, including the position defined therein and the upstream and / or downstream parts of the position.

[0086] The “at least one” mentioned above refers to any one, any combination of two, any combination of three, ..., or all combinations of all the types defined therein, and all are within the scope of protection of the present invention.

[0087] Among the amino acid sequences or nucleotide sequences involved in the above content, sequences with a homology of more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% with the sequences involved in the present invention, and / or sequences after substitution, deletion or insertion of amino acid residues or nucleotides on the basis of the sequences involved in the present invention, and sequences with the same or similar functions as the sequences involved in the present invention, are all within the scope of protection of the present invention.

[0088] Beneficial effects of the present invention

[0089] The present invention designs and synthesizes a guide RNA (sgRNA) that can guide the ABE protein to the target gene region and target the target sequence, and forms an RNP mixture with the ABE protein and electro-transduces it into CD34+ hematopoietic stem / progenitor cells (HSPCs) derived from β-thalassemia patients, thereby achieving efficient single editing of the promoter region of the HBG1 / 2 gene or the enhancer region of the BCL11A gene, or co-editing of the promoter region of the HBG1 / 2 gene and the enhancer region of the BCL11A gene, or simultaneous editing of the promoter region of the HBG1 / 2 gene and the enhancer region of the BCL11A gene and the transferrin receptor gene TFR2; thereby activating or enhancing the expression of the γ-globin gene by editing the HBG1 gene promoter and the BCL11A gene enhancer, and promoting the formation of red blood cells with normal function by knocking out or knocking down the expression of the transferrin receptor TFR2 by editing.

[0090] The present invention can use single-base editing technology to edit HSPCs from patients with transfusion-dependent β-thalassemia and sickle cell anemia with high editing efficiency; the edited autologous HSPCs are returned to the patient's body to rebuild the entire blood system and differentiate into healthy red blood cells with normal physiological functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 Sanger sequencing plots of ABE8e editing efficiency at the sgHBGsense site in healthy human hematopoietic stem / progenitor cells. The left image shows editing with sgHBGsense alone, the middle image shows co-editing with sg1620 and sgHBGsense, and the right image shows an unedited control.

[0092] Figure 2 Deep sequencing test results of the sgHBGsense site editing efficiency when the sgHBGsense site is edited alone in healthy human hematopoietic stem / progenitor cells, or when the sg1620 site is co-edited with sgHBGsense.

[0093] Figure 3 These are the deep sequencing test results of the editing efficiency of ABE8e when eight sgRNA sites, including sgHBGsense, sgHBGsite1, sgHBGnspacer1, sgHBGnspacer2, sgHBGnspacer3, sgHBGnspacer4, sgHBGnspacer5, and sgHBGnspacer6, are individually edited in the HBG1 promoter region of healthy human hematopoietic stem / progenitor cells.

[0094] Figure 4Sanger sequencing plots of the editing efficiency of ABE7.10 and ABE8e at the sg1620 site in healthy human hematopoietic stem / progenitor cells. From left to right, the first plot shows the editing results of ABE7.10 at the sg1620 site; the second plot shows the editing results of ABE8e editing the sg1620 site alone; the third plot shows the editing results of ABE8e co-editing the sg1620 and sgHBGsense sites at the sg1620 site; and the fourth plot shows the sequencing spectrum of the unedited control at the sg1620 site.

[0095] Figure 5 Deep sequencing test results of the editing efficiency of the sg1620 site when the sg1620 site was edited alone in healthy human hematopoietic stem / progenitor cells, or when the sg1620 site was co-edited with sgHBGsense.

[0096] Figure 6 These are the deep sequencing test results of the editing efficiency of ABE8e when five sgRNA sites, including sg1620, sg1617, sg1618, sg1619, and sg1621, are individually edited in the BCL11A enhancer region of healthy human hematopoietic stem / progenitor cells.

[0097] Figure 7 ABE8e was used to introduce mutations in the BCL11A enhancer region of healthy human hematopoietic stem / progenitor cells at five sgRNA sites, including sg1620, sg1617, sg1618, sg1619, and sg1621. The cells were differentiated in vitro for 18 days, and q-PCR was used to detect changes in HbF levels caused by base editing.

[0098] Figure 8 Sanger sequencing of ABE8e editing efficiency at the TFR2_Nontrans_sg1 locus in hematopoietic stem / progenitor cells from patients with β-thalassemia. The left panel shows editing with TFR2_Nontrans_sg1 alone, the middle panel shows co-editing with sg1620, sgHBGsense, and TFR2_Nontrans_sg1, and the right panel shows an unedited control.

[0099] Figure 9 Deep sequencing test results of the editing efficiency of the TFR2_Nontrans_sg1 site when the TFR2_Nontrans_sg1 site is edited alone in hematopoietic stem / progenitor cells of patients with β-thalassemia, or when the sg1620 site, sgHBGsense and TFR2_Nontrans_sg1 site are co-edited.

[0100] Figure 10After ABE8e edited the TFR2_Nontrans_sg1 targeted site in the hematopoietic stem / progenitor cells of β-thalassemia patients, Western blot experiments were performed to detect the expression results of TFR2 protein.

[0101] Figure 11 ABE8e was introduced into the sg1620 site, sgHBG sense site, or TFR2_Nontrans_sg1 of hematopoietic stem / progenitor cells from β-thalassemia patients by editing them individually or co-editing. The cells were differentiated in vitro for 18 days, and q-PCR was used to detect changes in γ-globin mRNA levels caused by base editing.

[0102] Figure 12 ABE8e was introduced into healthy human hematopoietic stem / progenitor cells by co-editing the sg1620 site with sgHBGnspacer1, sgHBGnspacer2, sgHBGnspacer3, sgHBGnspacer4, sgHBGnspacer5, and sgHBGnspacer6 to form six combinations. The cells were differentiated in vitro for 18 days, and q-PCR was used to detect changes in γ-globin mRNA levels caused by base editing.

[0103] Figure 13 Sanger sequencing plots of the editing efficiency of ABE8e at the 55_sgRNA1 and 55_sgRNA2 sites in the +55kb enhancer region of the BCL11A gene in healthy human hematopoietic stem / progenitor cells. The left panel shows editing using the ABE8e-55_sgRNA1 combination, the middle panel shows editing using the ABE8e-55_sgRNA2 combination, and the right panel shows an unedited control.

[0104] Figure 14 Figures show the Sanger sequencing results and quantitative analysis of the editing efficiency of ABE8e at the 55_sgRNA1 and 55_sgRNA2 sites in the +55kb enhancer region of the BCL11A gene in healthy human hematopoietic stem / progenitor cells, using the EditR method (EditR is available at baseEditR.com / and https: / / github.com / MoriarityLab / EditR7). The top figure shows the Sanger sequencing spectrum for editing using the ABE8e-55_sgRNA1 combination, the middle figure shows the Sanger sequencing spectrum for editing using the ABE8e-55_sgRNA2 combination, and the bottom figure shows the unedited control. The right figure shows a bar chart of the statistical editing efficiency.

[0105] Figure 15ABE8e was used to introduce mutations at the 55_sgRNA1 site and the 55_sgRNA2 site in the +55kb enhancer region of the BCL11A gene in healthy human hematopoietic stem / progenitor cells. The cells were differentiated in vitro for 18 days, and qPCR was used to detect changes in the transcription level of the γ-globin gene caused by base editing.

[0106] Figure 16 ABE8e was used to introduce mutations at the 55_sgRNA1 site and the 55_sgRNA2 site in the +55kb enhancer region of the BCL11A gene in healthy human hematopoietic stem / progenitor cells. The cells were differentiated in vitro for 18 days, and HPLC was used to detect changes in γ-globin levels caused by base editing.

[0107] Figures 17A to 17D Figure 3. Editing efficiency of ABE8e at the 55_sgRNA1 site in the +55kb enhancer region of the BCL11A gene, the sg1620 site in the +58kb enhancer region of the BCL11A gene, and the sgHBGsense site in the enhancer region of the HBG1 / 2 genes, either alone or with the dual editing combinations of 55_sgRNA1+sg1620 and 55_sgRNA1+HBGsense in healthy human hematopoietic stem / progenitor cells. Figure a shows the Sanger sequencing efficiency of the 55_sgRNA1 site when editing the site targeted by 55_sgRNA1 alone. Figure b shows the Sanger sequencing efficiency of the 55_sgRNA1 site when co-editing with 55_sgRNA1 and sg1620. Figure c shows the Sanger sequencing efficiency of the 55_sgRNA1 site when co-editing with 55_sgRNA1 and sgHBGsense. Figure d shows an unedited control mock.

[0108] Figure e shows the Sanger sequencing efficiency of the sgRNA1620 site when the site targeted by sgRNA1620 is edited alone. Figure f shows the Sanger sequencing efficiency of the sgRNA1620 site when 55_sgRNA1 and sg1620 are co-edited. Figure g shows an unedited control mock.

[0109] Figure h is the Sanger sequencing spectrum of the editing efficiency of the HBGsense site when the site targeted by sgHBGsense is edited alone, Figure i is the Sanger sequencing spectrum of the editing efficiency of the sgHBGsense site when 55_sgRNA1 and sgHBGsense are co-edited, and Figure j is the unedited control Mock.

[0110] Figure 18When ABE8e was used to perform single editing, double editing combination, or triple-site co-editing at the 55_sgRNA1 site, sg1620 site, or sgHBGsense site in healthy human hematopoietic stem / progenitor cells, the cells were differentiated in vitro for 18 days, and qPCR was used to detect changes in γ-globin gene transcription levels caused by base editing.

[0111] Figure 19 When ABE8e was edited single or double or co-edited at the 55_sgRNA1 site, sg1620 site, or sgHBGsense site in healthy human hematopoietic stem / progenitor cells, the cells were differentiated in vitro for 18 days and the changes in γ-globin levels caused by base editing were detected by HPLC. DETAILED DESCRIPTION

[0112] The present invention will be described in further detail below in conjunction with specific examples and accompanying drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, variations and advantages that can be imagined by those skilled in the art are included in the present invention, and are protected by the appended claims. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no special restrictions. As recorded in Sambrook et al., Molecular Cloning, A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the recommended conditions of the manufacturer.

[0113] The construction method of the present invention for introducing A>G substitution at different target sites in human hematopoietic stem / progenitor cells comprises the following steps:

[0114] (1) sgRNA design;

[0115] (2) sgRNA synthesis and ABE8e protein preparation;

[0116] (3) mixing the above-mentioned sgRNA and ABE8e protein in a molar ratio of (1-3):1 and electroporating them into human hematopoietic stem / progenitor cells;

[0117] (4) The electroporated cells were cultured in a CD34+EDM-1 differentiation system for 7 days. 96 hours after electroporation, the genomic DNA of the cells obtained in the above step was extracted, and PCR amplification was performed and sent for Sanger sequencing or deep sequencing to determine the mutation efficiency;

[0118] (5) After the mutation is confirmed, the cells are differentiated into EDM-2 for 4 days and EDM-3 for 7 days. During the differentiation process, appropriate time points are selected to detect the expression of corresponding protein mRNA, such as Western blot detection of TFR2 protein expression in the early stage of differentiation; after the differentiation is completed, RNA is extracted from a portion of cells, reverse transcribed into cDNA, and real-time fluorescence quantitative PCR (qPCR) is used to detect globin mRNA expression; after the differentiation is completed, another portion of cells is lysed with ice water to obtain the supernatant, and the level of γ-globin or HbF protein is detected by high-performance liquid chromatography (HPLC).

[0119] The hematopoietic stem / progenitor cells used in the present invention include hematopoietic stem / progenitor cells from healthy individuals and hematopoietic stem / progenitor cells from patients with β-thalassemia (CD34+ HSPCs). The chemically modified sgRNAs are electroporated into healthy human hematopoietic stem / progenitor cells or β-thalassemia hematopoietic stem / progenitor cells, either alone or in combination, with the ABE8e protein to form a mixture. This allows for efficient introduction of A>G substitutions, either alone or simultaneously, at one or more gene target sites in hematopoietic stem / progenitor cells from healthy individuals or patients with β-thalassemia, thereby achieving separate or co-editing of the promoter region of the HBG1 gene and the enhancer region of the BCL11A gene, which are associated with γ-globin expression, to activate or enhance the expression of fetal hemoglobin HbF (HbF: α2γ2). Alternatively, co-editing of the transferrin receptor gene TFR2 can be performed to knock out or reduce the expression of the transferrin receptor TFR2, thereby promoting the production of normally functioning red blood cells and treating β-thalassemia (β-thalassemia) and sickle cell anemia (sickle cell anemia).

[0120] Unless otherwise specified in the following examples, the hematopoietic stem / progenitor cells used were derived from patients with β-thalassemia.

[0121] Example 1: Using an adenine base editor (ABE) to edit the promoter region of the HBG1 / 2 gene encoding gamma globulin in healthy human hematopoietic stem / progenitor cells (using electroporation as an example)

[0122] To mimic natural HPFH and SNPs, we used ABE base editors to target the introduction of A>G or T>C base mutations in the relevant regions, thereby reactivating the γ-globin encoding gene and maintaining functional hemoglobin HbF synthesis in the adult stage.

[0123] 1. sgRNA design

[0124] sgRNAs were designed within the range of -90 bp to -220 bp of the promoter region of the HBG1 gene and the range of -90 bp to -220 bp of the promoter region of the HBG2 gene. They were numbered sgHBGsense, sgHBGsite1, sgHBGnspacer1, sgHBGnspacer2, sgHBGnspacer3, sgHBGnspacer4, sgHBGnspacer5, and sgHBGnspacer6, and their target sequences were:

[0125] sgHBGsense: 5′-CTTGACCAATAGCCTTGACA-3′ (SEQ ID No: 1, located at positions −120 to −101 in the promoter region of HBG1 / 2 genes);

[0126] sgHBGsite1: 5′-CTTGTCAAGGCTATTGGTCA-3′ (SEQ ID No: 2, reverse complementary sequence located at positions −118 to −99 of the promoter region of HBG1 / 2 genes);

[0127] sgHBGnspacer1: 5′-ATGCAAATATCTGTCTGAAA-3′ (SEQ ID No: 3, located at positions −182 to −163 of the promoter region of the HBG1 / 2 gene);

[0128] sgHBGnspacer2: 5′-ATATTTGCATTGAGATAGTG-3′ (SEQ ID No: 4, reverse complementary sequence located at positions −192 to −173 of the promoter region of the HBG1 / 2 gene);

[0129] sgHBGnspacer3: 5′-TATTTGCATTGAGATAGTGT-3′ (SEQ ID No: 5, reverse complementary sequence located at positions −193 to −174 of the promoter region of the HBG1 / 2 genes);

[0130] sgHBGnspacer4: 5′-ATTTGCATTGAGATAGTGTG-3′ (SEQ ID No: 6, reverse complementary sequence located at positions −194 to −175 of the promoter region of the HBG1 / 2 gene);

[0131] sgHBGnspacer5: 5′-GCATTGAGATAGTGTGGGGA-3′ (SEQ ID No: 7, reverse complementary sequence located at positions −198 to −179 of the promoter region of the HBG1 / 2 gene);

[0132] sgHBGnspacer6: 5′-GTGGGGAAGGGGCCCCCAAG-3′ (SEQ ID No: 8, reverse complementary sequence located at positions −211 to −192 of the promoter region of the HBG1 / 2 gene);

[0133] HBG1 / 2 gene represents HBG1 gene and / or HBG2 gene.

[0134] 2. Preparation of sgRNA and ABE8e protein

[0135] In step 1 of chemical modification synthesis, a total of 8 sgRNAs were synthesized, and ABE8e protein (whose amino acid sequence is shown in SEQ ID No: 25) was prepared simultaneously.

[0136] 3. Obtain recombinant cells by electroporation

[0137] Mix any of the sgRNAs in step 2 with ABE8e protein in a certain ratio (2:1) and incubate at room temperature for 10 min to obtain eight sgRNA and ABE8e protein complexes (RNPs). Electroporate any of the eight RNPs into healthy human hematopoietic stem / progenitor cells (the ratio of ABE8e and sgRNA complex to cells is 30 μg complex: 1×10 4 cells), and healthy human hematopoietic stem / progenitor cells without any RNP added were used as blank controls. The electroporation solution was mixed according to the ratio of the electroporation kit, and the number of electroporated cells did not exceed 10. 5 After cell centrifugation, the cells were resuspended in electroporation solution and gently mixed with the incubated RNPs, and then transferred to an electroporation cup. Avoid generating bubbles during the operation, and use the CD34 cell electroporation program EO-100 for electroporation (Lonza-4D electroporator). After confirming that the electroporation was successful, the cells were incubated at room temperature for 5 minutes, re-centrifuged to remove ABE8e protein and electroporation solution, and the cells were resuspended in CD34+EDM-1 medium and added to a cell culture plate for differentiation culture at 37° to obtain recombinant cells, i.e., recombinant hematopoietic stem / progenitor cells, wherein the RNPs were guided to the target site by sgRNA, and an A>G substitution was introduced within the editing window of ABE8e at the target site.

[0138] 4. Identification results

[0139] (1) Sanger sequencing to identify mutations in genomic DNA

[0140] Methods: After the recombinant hematopoietic stem / progenitor cells prepared in step 3 were cultured in vitro for 4 days, an appropriate amount of cells were collected and genomic DNA was extracted. After PCR amplification, the mutation efficiency was detected by Sanger sequencing. The primer sequences used for PCR at the sgHBGsense site were as follows:

[0141] Test115F: 5'-TACAGGCCTCACTGGAGCTA-3' (SEQ ID NO: 26);

[0142] Test115R: 5'-GAAGCGACCTGGACTTTTGC-3' (SEQ ID NO: 27).

[0143] Results: As Figure 1 As shown, compared to the unedited blank control (Mock), in edited hematopoietic stem cells, ABE8e successfully introduced an A>G mutation within the editing window when editing the site targeted by sgHBGsense alone. The sites where the mutations were introduced are marked with dotted black boxes. Within the black boxes, it is clearly visible that the A peaks at different sites are mutated to G peaks at varying efficiencies. Furthermore, the A to G conversion at the +5A and +8A positions of sgHBGsense is significant, with the G peak height even far exceeding the A peak height, preliminarily indicating high editing efficiency at this site.

[0144] (2) Deep sequencing to identify mutations in genomic DNA

[0145] Methods: After the recombinant hematopoietic stem / progenitor cells prepared in step 3 were cultured in vitro for 4 days, an appropriate amount of cells were collected and genomic DNA was extracted. After PCR amplification, deep sequencing was performed to detect the mutation efficiency. The primer sequences used for PCR at the sgHBGsense site were as follows:

[0146] Deepseq-HBG115-tryF: 5'-CCCCTTCCCCACACTATCTCA-3' (SEQ ID NO: 28);

[0147] Deepseq-HBG115-tryR: 5'-ATTCTTCATCCCTAGCCAGCC-3' (SEQ ID NO: 29).

[0148] Results: As Figure 2 As shown, the editing efficiency of ABE8e at the sites of the HBG1 / 2 genes targeted by sgHBGsense is: +5A>G 60-70%, +8A>G 70-80%, +9A>G 10-20%, and +11A>G 40-50%. Among them, the A at positions +8, +9, and +11 all fall within the range that can maintain a natural △13bp HPFH mutation in the HBG1 gene promoter region. It is reported that this △13bp HPFH mutation can maintain 30-32% HbF expression in adults. Therefore, the high-efficiency mutations introduced by ABE8e are expected to have the effect of enhancing HbF expression.

[0149] In addition, the editing efficiency of ABE8e at the sites of HBG1 / 2 genes targeted by sgHBGsite1 was: +7A>G 93.2%, +8A>G 27.0%;

[0150] The editing efficiency of ABE8e at the sites of HBG1 / 2 genes targeted by sgHBGnspacer1 was: +5A>G 67.8%, +6A>G 55.2%, +7A>G 50.3%, and +9A>G 53.9%;

[0151] The editing efficiency of ABE8e at the sites of HBG1 / 2 genes targeted by sgHBGnspacer2 was: +3A>G 12.8%, +9A>G 14.8%;

[0152] The editing efficiency of ABE8e at the site of HBG1 / 2 gene targeted by sgHBGnspacer3 is: +8A>G 67.6%;

[0153] The editing efficiency of ABE8e at the site of HBG1 / 2 gene targeted by sgHBGnspacer4 is: +7A>G 78.3%;

[0154] The editing efficiency of ABE8e at the sites of HBG1 / 2 genes targeted by sgHBGnspacer5 was: +3A>G 34.3%, +7A>G 40.1%, +9A>G 6.4%, and +11A>G 34.8%;

[0155] The editing efficiency of ABE8e at the sites of HBG1 / 2 genes targeted by sgHBGnspacer6 was: +7A>G 22.7%, +8A>G 15.1%;

[0156] The editing efficiency of ABE8e at the sites of HBG1 / 2 genes targeted by the above 8 sgRNAs is as follows Figure 3 shown.

[0157] (3) EDM-2 and EDM-3 differentiation culture

[0158] After sequencing confirms that the target site mutation is successful, the cells can continue to differentiate in EDM-2 medium after completing the EDM-1 stage. After the EDM-2 differentiation stage is completed, the cells are transferred to EDM-3 for further differentiation. After the differentiation is completed, RNA is extracted and reversed into cDNA for use in qPCR experiments to detect γ-globin expression.

[0159] Example 2: Using an adenine base editor (ABE) to edit the enhancer region of the BCL11A gene, a transcriptional repressor of the γ-globin gene, in healthy human hematopoietic stem / progenitor cells (using electroporation as an example)

[0160] 1. sgRNA design

[0161] sgRNAs were designed in the enhancer +58kb region of BCL11A, numbered sg1617, sg1618, sg1619, sg1620, and sg1621, and their target sequences were:

[0162] sg1617: 5′-CTAACAGTTGCTTTTATCAC-3′ (SEQ ID No: 9, located at positions 60495264-60495283 of human chromosome 2);

[0163] sg1618: 5′-TTGCTTTTATCACAGGCTCC-3′ (SEQ ID No: 10, located at positions 60495257-60495276 of human chromosome 2);

[0164] sg1619: 5′-TTTTATCACAGGCTCCAGGA-3′ (SEQ ID No: 11, located at positions 60495253-60495272 of human chromosome 2);

[0165] sg1620: 5′-TTTATCACAGGCTCCAGGAA-3′ (SEQ ID No: 12, located at positions 60495252-60495271 of human chromosome 2);

[0166] sg1621: 5′-CACAGGCTCCAGGAAGGGTT-3′ (SEQ ID No: 13, located at positions 60495247-60495266 of human chromosome 2);

[0167] 2. Preparation of sgRNA, ABE8e protein, and ABE7.10 protein

[0168] In step 1 of chemical modification synthesis, a total of 5 sgRNAs were prepared, and ABE8e protein (whose amino acid sequence is shown in SEQ ID No: 25) and ABE7.10 protein (whose amino acid sequence is as described in the literature "Koblan, LW, et al., Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol, 2018. 36(9): p. 843-846.") were prepared simultaneously.

[0169] 3. Obtain recombinant cells by electroporation

[0170] Proceed according to the method of step 3 of Example 1.

[0171] 4. Identification results

[0172] (1) Sanger sequencing to identify mutations in genomic DNA

[0173] Method: The method in step 4 (1) of Example 1 was followed, wherein the primer sequences used for PCR at the sg1620 site were as follows:

[0174] 58-checksimilar-F: 5'-AGCATCACAACAGGCAGAGAAT-3' (SEQ ID NO: 30);

[0175] 58-checksimilar-R: 5'-GGGAACACAGATCCTAACACAGT-3' (SEQ ID NO: 31).

[0176] Results: As Figure 4 As shown, a comparison of the efficiency of ABE7.10 and ABE8e reveals that ABE7.10 has almost no editing efficiency in hematopoietic stem / progenitor cells, while ABE8e has significantly higher editing efficiency than ABE7.10. Compared to an unedited control mock, ABE8e alone successfully introduced an A>G mutation within the editing window of the site targeted by the sgRNAs used when editing the site targeted by sg1620. The sites where the mutations were introduced are marked with dashed black boxes. Within the black boxes, it is clearly visible that the A peaks at different sites are mutated to G peaks with varying efficiencies. Furthermore, the A-to-G conversion at the +4A and +7A positions of sg1620 is significant, with the G peak height even far exceeding the A peak height, preliminarily indicating high editing efficiency at these sites.

[0177] (2) Deep sequencing to identify mutations in genomic DNA

[0178] Method: The method in step 4 (2) of Example 1 was followed, wherein the primer sequences used for PCR at the sg1620 site were as follows:

[0179] DeepSPCR-F-try: 5'-GCCAGAAAAGAGATATGGCATC-3' (SEQ ID NO: 32);

[0180] DeepSPCR-R-try: 5'-AGAGAGCTTCCGAAAGAGG-3' (SEQ ID NO: 33).

[0181] Results: As Figure 5As shown, the positions and efficiencies of the A>G mutations introduced at the sites targeted by sg1620 are: +4A>G 70-80%, +7A>G 90-100%, and +9A>G 20-30%. The three edited A positions at the sg1620 site all fall within the semi-E-box / GATA1 motif at +58 of the DNase I hypersensitive site (DHS) in the enhancer region of the BCL11A gene. Therefore, the high-efficiency mutations introduced by ABE8e are expected to disrupt the binding of transcription factors to this region and enhance HbF expression.

[0182] In addition, we also tested the efficiency of other sgRNAs designed in the enhancer region of the BCL11A gene. The editing efficiency of ABE8e at the sites in the enhancer region of BCL11A targeted by sg1617 was: +3A>G 5.4%, +4A>G 15.7%, and +6A>G 47.5%;

[0183] The editing efficiency of ABE8e at the sites in the enhancer region of BCL11A targeted by sg1618 is: +9A>G 28.6%, +12A>G 28.4%, +14A>G 12.5%;

[0184] The editing efficiency of ABE8e at the sites in the enhancer region of BCL11A targeted by sg1619 is: +5A>G 82.8%, +8A>G 67.4%, +10A>G 31.4%;

[0185] The editing efficiency of ABE8e at the sites in the enhancer region of BCL11A targeted by sg1621 is: +2A>G 64.0%, +4A>G 20.5%;

[0186] The editing efficiency of ABE8e at the sites in the BCL11A gene enhancer region targeted by the above five sgRNAs is as follows: Figure 6 shown.

[0187] (3) EDM-2 and EDM-3 differentiation culture

[0188] Proceed according to the method in step 4 (3) of Example 1.

[0189] In addition, the editing effect of ABE8e at the sites of the above five sgRNAs targeting the BCL11A gene enhancer region of healthy human hematopoietic stem / progenitor cells is as follows Figure 7As shown. The results showed that, compared to the unedited control mock, ABE8e editing at sites sg1618, sg1619, sg1620, and sg1621, in addition to sg1617, increased γ-globin mRNA levels to varying degrees. ABE8e editing at sites sg1619 and sg1620 significantly increased γ-globin mRNA levels, exceeding 40%. This is consistent with previous research findings: the A position edited at the sg1617 site falls outside the half-E-box / GATA1 motif (transcription factor binding region) at the DNase I hypersensitive site (DHS) +58 in the enhancer region of the BCL11A gene. Therefore, the mutation introduced by ABE8e does not disrupt transcription factor binding to this region and is therefore ineffective in increasing γ-globin mRNA expression levels. The edited A positions at sites sg1618, sg1619, sg1620, and sg1621 all fall within the semi-E-box / GATA1 motif at the DNase I hypersensitive site (DHS) +58 in the enhancer region of the BCL11A gene. Therefore, the high-efficiency mutation introduced by ABE8e indeed disrupts transcription factor binding to this region and increases γ-globin mRNA expression. These results from healthy individuals suggest that if the four RNP combinations of ABE8e-sg1618, ABE8e-sg1619, ABE8e-sg1620, and ABE8e-sg1621 are applied to β-thalassemia hematopoietic stem / progenitor cells using the method described in step 3 of Example 1, each of these RNP combinations will have the effect of increasing HbF levels. In particular, the ABE8e-sg1619 and ABE8e-sg1620 combinations have the potential to significantly increase HbF levels and alleviate thalassemia symptoms.

[0190] Example 3: Editing the gene coding region and intron region of the transferrin receptor TFR2 in β-thalassemia hematopoietic stem / progenitor cells using an adenine base editor (ABE) (using electroporation as an example)

[0191] Various studies have found that iron restriction can help alleviate erythropoiesis disorders in mouse models of β-thalassemia. Artuso et al. further demonstrated that transplanting TFR2-knockout bone marrow in β-thalassemia mice increased hemoglobin levels and the proportion of mature erythroid progenitor cells in the bone marrow.

[0192] Therefore, this embodiment uses two methods to achieve the knockout or knockdown expression of human hematopoietic stem cell TFR2, thereby improving the red blood cell production of patients with thalassemia and achieving the effect of disease treatment. Method one, using ABE protein, mutates the "A" in the translation initiation region "ATG" of the gene to "G", thereby destroying protein translation and achieving the purpose of knocking out gene expression; Method two, using ABE protein in the intron splicing receiving region - "AG" (acceptor), edits the "A" in "AG" to "G", thereby causing splicing problems, resulting in abnormal protein expression, and thus destroying protein function. Preferably, method one.

[0193] 1. sgRNA design

[0194] Five sgRNAs were designed within the translation initiation codon (ATG) region of the TFR2 gene (NCBI Gene ID: 7036) and the five non-frameshift ATGs following it and their surrounding regions. Their numbers and target sequences are as follows:

[0195] TFR2_Nontrans_sg1: 5'-ACAAGCATGGAGCGGCTTTG-3' (SEQ ID No: 16);

[0196] TFR2_Nontrans_sg2: 5'-CAGCTCCATGGGGCAGAAGT-3' (SEQ ID No: 17);

[0197] TFR2_Nontrans_sg3: 5'-GGCCATGTTCCTGCAGTTCC-3' (SEQ ID No: 18);

[0198] TFR2_Nontrans_sg4: 5'-CGGGATGGCCGCTCTGACTC-3' (SEQ ID No: 19);

[0199] TFR2_Nontrans_sg5: 5'-CTCCATGGTGAGCAACGGTA-3' (SEQ ID No: 20);

[0200] The target sequences of the five sgRNAs designed above are all located in the human TFR2 gene.

[0201] Four sgRNAs were designed within the intronic splicing acceptor region of the TFR2 gene, "AG" (acceptor). Their numbers and target sequences are as follows:

[0202] TFR2_ABEskip_sg1: 5'-GCTTAGGCAAACCAGCCTTC-3' (SEQ ID No: 21);

[0203] TFR2_ABEskip_sg2: 5'-TCCCCAGGTGACCAATGCTC-3' (SEQ ID No: 22);

[0204] TFR2_ABEskip_sg3: 5'-CTGCAGGTGCACCTGGGAAC-3' (SEQ ID No: 23);

[0205] TFR2_ABEskip_sg4: 5'-CAGGATCCGGCCCCTACCCA-3' (SEQ ID No: 24);

[0206] The target sequences of the four sgRNAs designed above are all located in the human TFR2 gene.

[0207] 2. Preparation of sgRNA and ABE8e protein

[0208] TFR2_Nontrans_sg1 in step 1 was chemically modified and synthesized, and ABE8e protein (whose amino acid sequence is shown in SEQ ID No: 25) was prepared simultaneously.

[0209] 3. Obtain recombinant cells by electroporation

[0210] Proceed according to the method of step 3 of Example 1.

[0211] 4. Identification results

[0212] (1) Sanger sequencing to identify mutations in genomic DNA

[0213] Method: The method in step 4 (1) of Example 1 was followed, wherein the Sanger sequencing PCR primer sequences for the TFR2_Nontrans_sg1 site were as follows:

[0214] First round PCR primers:

[0215] Nontrans_PCR-F5: 5'-GGGAACTAGGAGGCCAAAGT-3' (SEQ ID NO: 34);

[0216] Nontrans_PCR-R2: 5'-TGGGCATGAGATTGGGGCAA-3' (SEQ ID NO: 35);

[0217] Second round PCR primers:

[0218] Nontrans_PCR-F3: 5'-GTCCGCCCCAAGGTCAAAAA-3' (SEQ ID NO: 36);

[0219] Nontrans_PCR-R5: 5'-AGGCATCTGGCAATAATGAGGT-3' (SEQ ID NO: 37).

[0220] Results: As Figure 8 As shown, compared to an unedited control mock, when editing the TFR2_Nontrans_sg1-targeted site, the edited hematopoietic stem cell ABE8e successfully introduced an A>G mutation within the editing window of the site targeted by the sgRNA. The sites where the mutations were introduced are marked with dashed black boxes. Within the black boxes, it is clearly visible that the A peaks at different sites were mutated to G peaks at varying efficiencies. Furthermore, the A-to-G conversion at the TFR2_Nontrans_sg1+4A and +7A positions was significant, with the G peak height even far exceeding the A peak height, preliminarily indicating high editing efficiency at these sites.

[0221] (2) Deep sequencing to identify mutations in genomic DNA

[0222] Method: The method in step 4 (2) of Example 1 was followed, wherein the sequence of the PCR primer for deep sequencing of the TFR2_Nontrans_sg1 site was as follows:

[0223] TFR2nontrans-DSF1: 5'-ATCGCTGGGGGACAGCCTGC-3' (SEQ ID NO: 38);

[0224] TFR2nontrans-DSR2: 5'-CTCAGGGGCTTGGGAGGGGG-3' (SEQ ID NO: 39).

[0225] Results: As Figure 9 As shown, the editing efficiency of ABE8e at the site targeted by TFR2_Nontrans_sg1 is: +3A>G 50-60%, +4A>G 60-70%, +7A>G 90-100%; and the +7 position can almost 100% convert the start codon "ATG" to "GTG", and there is no suitable non-frameshifted new "ATG" in the spliced mature mRNA 100nt downstream of the start codon. Therefore, the high-efficiency mutation introduced by ABE8e is expected to disrupt protein translation initiation, thereby knocking down or even knocking out protein TFR2 expression.

[0226] (3) EDM-2 and EDM-3 differentiation culture

[0227] Proceed according to the method in step 4 (3) of Example 1.

[0228] (4) Western blot detection

[0229] Methods: After the recombinant hematopoietic stem / progenitor cells prepared in step 3 were differentiated and cultured in vitro for 3-4 days, an appropriate amount of cells were collected and total protein was extracted. TFR2-α and TFR2-β were detected by Western blot, using β-actin as an internal reference.

[0230] Results: As Figure 10 As shown, ABE8e protein was co-electroporated with TFR2_Nontrans_sg1, which could knock down TFR2-α expression.

[0231] Example 4: Using an adenine base editor (ABE), editing is performed individually or in combination within the promoter region of the γ-globin encoding gene HBG1 / 2, the enhancer + 58kb region of the transcriptional repressor BCL11A gene of the γ-globin encoding gene, and the translation start codon (ATG) region of the TFR2 gene (NCBI Gene ID: 7036) and its surrounding area in β-thalassemia hematopoietic stem / progenitor cells (using electroporation as an example).

[0232] 1. Obtain recombinant cells by electroporation

[0233] At least one sgRNA with a good editing effect was selected from Example 1 and recorded as sgRNA1. At least one sgRNA with a good editing effect was selected from Example 2 and recorded as sgRNA2. At least one sgRNA with a good editing effect was selected from Example 3 and recorded as sgRNA3. At least one sgRNA was selected from sgRNA1, sgRNA2, and sgRNA3 to form different sgRNA combinations. The sgRNA combinations were:

[0234] 1) sgHBGsense and sg1620;

[0235] 2) sgHBGsense, sg1620, and TFR2_Nontrans_sg1;

[0236] 3) sgHBGnspacer1 and sg1620;

[0237] 4) sgHBGnspacer2 and sg1620;

[0238] 5) sgHBGnspacer3 and sg1620;

[0239] 6) sgHBGnspacer4 and sg1620;

[0240] 7) sgHBGnspacer5 and sg1620;

[0241] 8) sgHBGnspacer6 and sg1620;

[0242] Any one of the sgRNA combinations 1) and 2) and the ABE8e protein were electroporated into β-thalassemia hematopoietic stem / progenitor cells according to the method in step 3 of Example 1, and β-thalassemia hematopoietic stem / progenitor cells without any RNP were used as a blank control to obtain recombinant hematopoietic stem / progenitor cells;

[0243] Any one of the sgRNA combinations 3)-8) and the ABE8e protein were electroporated into healthy human hematopoietic stem / progenitor cells according to the method in step 3 of Example 1, and healthy human hematopoietic stem / progenitor cells to which no RNP was added were used as blank controls to obtain recombinant hematopoietic stem / progenitor cells.

[0244] 2. Identification results

[0245] (1) Mutation identification of genomic DNA by Sanger sequencing

[0246] According to the corresponding method in Example 1-3, the results are as follows Figure 1 、 4 , 8. Compared with the control mock and editing with each sgRNA alone, double-site co-editing with sgHBGsense and sg1620, as well as triple-site co-editing with sgHBGsense, sg1620, and TFR2_Nontrans_sg1, successfully introduced A>G mutations within the editing window of the site targeted by each sgRNA contained in the RNP used.

[0247] (2) Deep sequencing to identify mutations in genomic DNA

[0248] According to the corresponding method in Example 1-3, the results are as follows Figure 2 、 5 , 9. Compared with editing by each sgRNA alone, the editing efficiency of the corresponding sites was at the same level when sgHBGsense and sg1620 were co-edited at two sites, and when sgHBGsense, sg1620, and TFR2_Nontrans_sg1 were co-edited at three sites. That is, there was no decrease in the efficiency of a single site when ABE8e co-edited two or three sites.

[0249] (3) qPCR detection

[0250] Methods: qPCR analysis was performed to analyze the mRNA expression of the γ-globin chain that constitutes HbF in hematopoietic stem cells edited in step 1, using single sgRNA editing as a control. The primer sequences used in PCR are as follows:

[0251] The qPCR primer sequences for γ-globin mRNA expression are:

[0252] HBG_γ-F: 5'-TGGGTCATTTCACAGAGGAG-3' (SEQ ID NO: 40);

[0253] HBG_γ-R: 5'-CATCTTCCACATTCACCTTGC-3' (SEQ ID NO: 41);

[0254] The qPCR primer sequences for β-globin mRNA expression are:

[0255] HBB_β-F: 5'-CAGTGCAGGCTGCCTATC-3' (SEQ ID NO: 42);

[0256] HBB_β-R: 5'-ATACTTGTGGGCCAGGGCAT-3' (SEQ ID NO: 43);

[0257] The qPCR primer sequences for α-globin mRNA expression are:

[0258] HBA_α-F: 5'-GCCCTGGAGAGGATGTTC-3' (SEQ ID NO: 44);

[0259] HBA_α-R: 5'-TTCTTGCCGTGGCCCTTA-3' (SEQ ID NO: 45);

[0260] The calculation formula is: HbF% = [γ / (γ+β)] × 100%, and the β / α value of the blank control is used as an internal reference.

[0261] result:

[0262] Compared with the unedited control group Mock, there was no significant difference in β / α in the edited cells.

[0263] like Figure 11As shown, ABE8e editing alone at sg1620 and sgHBGsense effectively increased HbF expression, restoring γ-globin mRNA levels by 55.7% and 64.5%, respectively. Co-editing with ABE8e at both sg1620 and sgHBGsense targets was even more pronounced, restoring γ-globin mRNA levels to 85.1%. Furthermore, co-editing with ABE8e at sg1620, sgHBGsense, and TFR2_Nontrans_sg1 also demonstrated significant efficacy compared to the control, restoring γ-globin mRNA levels by up to 75.3%. Previous studies have shown that HbF levels exceeding 30% represent the threshold for effective remission in SCD. Therefore, co-editing with sg1620 and sgHBGsense—that is, editing the BCL11A enhancer and HBG promoter regions in patients' hematopoietic stem / progenitor cells—can effectively alleviate thalassemia symptoms.

[0264] like Figure 12 As shown, the HbF level of cells edited by any of the sgRNA combinations 3)-8) and the ABE8e protein was significantly improved compared to the unedited control Mock. Among them, the HbF level of cells edited by the four combinations of sg1620+sgHBGnspacer2, sg1620+sgHBGnspacer3, sg1620+sgHBGnspacer4, and sg1620+sgHBGnspacer6 can reach 30-40%. This result in healthy people suggests that if the RNPs of these four combinations are applied to β-thalassemia hematopoietic stem / progenitor cells using the method of step 3 in Example 1, these four combinations of RNPs will have the potential to significantly increase HbF levels and alleviate the symptoms of thalassemia.

[0265] This example further verifies that: using an adenine base editor (ABE) to introduce an A>G mutation at the target sites in the promoter region of the HBG1 / 2 gene and the enhancer region of the BCL11A gene, thereby activating or enhancing γ-globin expression, replacing the defective β-globin function in patients with thalassemia and sickle cell anemia; and using ABE to introduce an A>G mutation in the start translation codon (ATG) region and the intron splicing donor site region of the TFR2 gene, resulting in the knockout / knockdown of TFR2 protein expression, will promote the sensitivity of erythroid progenitor cells to erythropoietin, reduce the apoptosis of erythroid precursor cells, promote erythropoiesis in patients with β-thalassemia, and is safe and feasible.

[0266] Example 5: Using an adenine base editor (ABE) to edit the enhancer +55kb region of the BCL11A gene, a transcriptional repressor encoding the γ-globin gene, in healthy human hematopoietic stem / progenitor cells (using electroporation as an example)

[0267] 1. sgRNA design

[0268] sgRNAs were designed in the region of BCL11A enhancer + 55kb from the transcription start site, named 55_sgRNA1 and 55_sgRNA2, respectively, and their target sequences were:

[0269] 55_sgRNA1: 5′-CACTGATAGGGGTCGCGGTA-3′ (SEQ ID No: 14, located at positions 60498359-60498378 of human chromosome 2);

[0270] 55_sgRNA2: 5′-GCACTGATAGGGGTCGCGGT-3′ (SEQ ID No: 15, located at positions 60498360-60498379 of human chromosome 2);

[0271] 2. Preparation of sgRNA and ABE8e protein

[0272] In step 1 of chemical modification synthesis, two sgRNAs were synthesized and ABE8e protein (the amino acid sequence of which is shown in SEQ ID No: 25) was prepared simultaneously.

[0273] 3. Obtain recombinant cells by electroporation

[0274] Follow the method of step 3 in Example 1.

[0275] 4. Identification results

[0276] (1) Sanger sequencing to identify mutations in genomic DNA

[0277] Methods: After the recombinant hematopoietic stem / progenitor cells prepared in step 3 were cultured in vitro for 4 days, an appropriate amount of cells were collected and genomic DNA was extracted. After PCR amplification, the mutation efficiency was detected by Sanger sequencing. The primer sequences used for PCR at the 55_sgRNA1 and 55_sgRNA2 sites were as follows:

[0278] DHS+55-F2: 5'-TTCATTGGCCGAGGATGACC-3' (SEQ ID NO: 46);

[0279] DHS+55-R2: 5'-ACCTCACTGGTTTCTCCCCT-3' (SEQ ID NO: 47).

[0280] Results: As Figure 13 As shown, compared to the unedited control mock, when editing the sites targeted by either 55_sgRNA1 or 55_sgRNA2, the edited hematopoietic stem / progenitor cells ABE8e successfully introduced A>G mutations within the editing window of the sgRNA-targeted sites. The sites where the mutations were introduced are marked with dashed black boxes. Within the black boxes, it is clearly visible that the A peaks at different sites were mutated to G peaks at varying efficiencies. Furthermore, the A peaks at +6A and +8A of 55_sgRNA1 were significantly converted to G peaks, with the G peak height far exceeding the A peak height. The A peaks at +3A, +7A, and +9A of 55_sgRNA2 were significantly converted to G peaks, preliminarily indicating high A>G editing efficiency at these sites.

[0281] (2) Quantitative analysis of Sanger sequencing results

[0282] Methods: Sanger sequencing files were analyzed and the mutation efficiency of target sites was quantified according to the following base editing efficiency analysis website:

[0283] https: / / hanlab.cc / beat / .

[0284] Results: As Figure 14 As shown, the sites targeted by 55_sgRNA1 introduced A>G mutations at a location and efficiency of 95% for +6A>G and 99% for +8A>G. The sites targeted by 55_sgRNA2 introduced A>G mutations at a location and efficiency of 17% for +3A>G, 52% for +7A>G, and 72% for +9A>G. Furthermore, the edited +6A and +8A positions of 55_sgRNA1, as well as the edited +7A and +9A positions of 55_sgRNA2, all fall within the GATA1 motif located +55 kb from the DNase I hypersensitive site (DHS) in the enhancer region of the BCL11A gene. Therefore, the high-efficiency A>G mutation introduced by ABE8e is expected to disrupt transcription factor binding to this region and enhance HbF expression.

[0285] (3) EDM-2 and EDM-3 differentiation culture

[0286] Once sequencing confirms the target site mutation, cells can continue differentiation in EDM-2 medium. After the EDM-2 differentiation stage is complete, cells are transferred to EDM-3 medium for further differentiation. After differentiation is complete, a portion of cells is extracted and converted into cDNA for qPCR analysis of γ-globin gene expression. Another portion of cells is shaken in ice water, disrupted, and centrifuged. The supernatant is collected and analyzed for γ-globin levels using high-performance liquid chromatography (HPLC).

[0287] qPCR test results are as follows Figure 15 The results showed that compared with the unedited control Mock, ABE8e editing at the 55_sgRNA1 and 55_sgRNA2 sites significantly increased γ-globin transcription levels to varying degrees. Among them, ABE8e editing at the 55_sgRNA1 site caused a more significant increase in γ-globin transcription, with the γ-globin mRNA level accounting for nearly 40% of the β-like globin mRNA (i.e., the sum of γ-globin and β-globin mRNA).

[0288] HPLC test results are as follows Figure 16 As shown. We used HPLC (DAD detector) to measure γ-globin levels in the ABE8e-55_sgRNA1 combination, which showed high γ-globin expression levels as detected by qPCR. The results showed that compared with the unedited control mock, editing of ABE8e at the 55_sgRNA1 site resulted in a significant increase in γ-globin levels, with the ratio of γ-globin to α-globin levels approaching 30%. Therefore, the above editing efficiency and editing effect in healthy subjects suggest that if the RNP of the ABE8e-55_sgRNA1 combination is applied to β-thalassemia hematopoietic stem / progenitor cells using the method of step 3 in Example 5, this combination of RNPs will have the potential to significantly increase HbF levels and alleviate TDT and SCD.

[0289] This example further verifies that an adenine base editor (ABE) is used to introduce an A>G mutation at the target site in the +55kb region of the BCL11A gene enhancer, thereby activating or enhancing γ-globin expression, replacing the defective globin in patients with thalassemia and sickle cell anemia, and promoting erythropoiesis in patients with β-thalassemia, and is safe and feasible.

[0290] Example 6: Using an adenine base editor (ABE) to edit the enhancer +55kb region, +58kb region, and promoter region of the HBG1 / 2 gene encoding the transcriptional repressor of the γ-globin gene in healthy human hematopoietic stem / progenitor cells (using electroporation as an example)

[0291] Based on the results of Example 5, we found that the high-efficiency A>G mutation introduced by ABE8e using 55_sgRNA1 in the +55kb region of the BCL11A gene enhancer significantly disrupted transcription factor binding to this region, thereby increasing HbF expression. Based on the results of Example 1, the high-efficiency A>G mutation introduced by ABE8e using sgHBGsense in the promoter region of the HBG1 / 2 genes activated or increased HbF expression. Based on the results of Example 2, the high-efficiency A>G mutation introduced by ABE8e using sg1619 or sg1620 in the +58kb region of the BCL11A gene enhancer activated or increased HbF expression. Therefore, in Example 6, we performed combined editing of any two or three regions within the +55kb region of the BCL11A gene enhancer, the +58kb region of the BCL11A gene enhancer, and the promoter region of the HBG1 / 2 genes. This is expected to have a more significant effect on increasing HbF expression than single-region editing.

[0292] 1. sgRNA design

[0293] Select 55_sgRNA1 in Example 5 in the region of BCL11A enhancer + 55 kb from the transcription start site;

[0294] sg1620 in Example 2 was selected in the region of the BCL11A enhancer +58 kb from the transcription start site;

[0295] The sgHBGsense in Example 1 was selected within the region including -90 bp to -220 bp of the promoter region of the HBG1 gene and within the region including -90 bp to -220 bp of the promoter region of the HBG2 gene.

[0296] 2. Preparation of sgRNA and ABE8e protein

[0297] Chemical modification synthesis: 55_sgRNA1 in Example 5, sg1620 in Example 2, and sgHBGsense in Example 1 were chemically synthesized to form three sgRNAs, and ABE8e protein (whose amino acid sequence is shown in SEQ ID No: 25) was prepared at the same time.

[0298] 3. Obtain recombinant cells by electroporation

[0299] The six combinations of 55_sgRNA1, sg1620, HBGsense, sg1620+sgHBGsense, 55_sgRNA1+sgHBGsense, 55_sgRNA1+sg1620, and 55_sgRNA1+sg1620+sgHBGsense in step 2 were mixed with ABE8e protein at a certain ratio (2:1) and incubated at room temperature for 10 min to obtain complexes (RNPs) formed by the six sgRNA combinations and ABE8e protein. Each RNP was electroporated into healthy human hematopoietic stem / progenitor cells (the ratio of ABE8e and sgRNA complex to cells was 30 μg complex: 1×10 4 cells), and healthy human hematopoietic stem / progenitor cells without any RNP added were used as blank control.

[0300] 4. Identification results

[0301] (1) Sanger sequencing to identify mutations in genomic DNA

[0302] Methods: After the recombinant hematopoietic stem / progenitor cells prepared in step 3 were differentiated and cultured in vitro for 4 days, an appropriate amount of cells were collected, and genomic DNA was extracted. After PCR amplification, the mutation efficiency was detected by Sanger sequencing. The primers used for PCR at the 55_sgRNA1 site were the same as those used in step 4 of embodiment 5; the primers used for PCR at the sg1620 site were the same as those used in step 4 of embodiment 2; and the primer sequences used for PCR at the sgHBGsense site were the same as those used in step 4 of embodiment 1.

[0303] Results: As Figure 17A As shown in Figures 1 and 2, compared to the unedited control mock, ABE8e successfully introduced an A>G mutation within the editing window of the site targeted by 55_sgRNA1 when editing the site targeted by 55_sgRNA1 alone or in combination with sg1620 or sgHBGsense for dual-site co-editing. The editing efficiency peaks at the site targeted by 55_sgRNA1 were essentially the same when editing alone and in combination. The sites where the mutations were introduced are marked with dotted black boxes. Within the black boxes, it is clearly visible that the A peaks at different sites are mutated to G peaks with varying efficiencies. Furthermore, the conversion of A peaks to G peaks at the +6A and +8A positions of 55_sgRNA1 is significant, with the G peak height being much higher than the A peak height, preliminarily indicating high A>G editing efficiency at these sites.

[0304] like Figure 17CAs shown, compared with the unedited control mock, ABE8e can successfully introduce A>G mutations within the editing window of the site targeted by sgRNA1620 when editing the site targeted by sgRNA1620 alone or in combination with 55_sgRNA1 for dual-site co-editing. There is basically no difference in the editing efficiency peak graphs at the site targeted by sgRNA1620 when editing alone and co-editing. The sites where mutations are introduced are marked with dotted black boxes. It can be clearly seen in the black boxes that the A peaks at different sites are mutated to G peaks with different efficiencies. In addition, the conversion of A peaks to G peaks at the +4A, +7A and +8A positions of sgRNA1620 is significant, and the G peak height is much higher than the A peak height, which preliminarily indicates that the A>G editing efficiency of these sites is high.

[0305] like Figure 17D As shown, compared with the unedited control Mock, when ABE8e edited the site targeted by sgHBGsense alone and combined with 55_sgRNA1 for dual-site co-editing, it was able to successfully introduce A>G mutations within the editing window of the site targeted by sgHBGsense. There was basically no difference in the peak graph of editing efficiency at the site targeted by sgHBGsense when editing alone and jointly. The sites where mutations were introduced are marked with dotted black boxes. It can be clearly seen in the black box that the A peaks at different sites are mutated to G peaks with different efficiencies. Moreover, the conversion of A peaks to G peaks at the +5A, +8A, +9A and +11A positions of HBGsense is significant, and the G peak height is much higher than the A peak height, which preliminarily indicates that the A>G editing efficiency of these sites is high.

[0306] (2) EDM-2 and EDM-3 differentiation culture

[0307] Once sequencing confirms the target site mutation, cells can continue differentiation in EDM-2 medium. After the EDM-2 differentiation stage is complete, cells are transferred to EDM-3 medium for further differentiation. After differentiation is complete, a portion of cells is collected for RNA extraction and conversion to cDNA for use in the following qPCR assay to detect γ-globin gene expression.

[0308] qPCR test results are as follows Figure 18The results showed that compared with the unedited control Mock, ABE8e was guided to the target site for editing by 55_sgRNA1, sg1620, sgHBGsense, sg1620+sgHBGsense, 55_sgRNA1+sg1620, 55_sgRNA1+sgHBGsense, and 55_sgRNA1+sg1620+sgHBGsense, and compared with the control Mock group, the γ-globin transcription level was increased to varying degrees; among them, ABE8e was guided to the target site for editing by 55_sgRNA1, sg1620, sgHBGsense, sg1620+sgHBGsense, 55_sgRNA1+sgHBGsense, 55_sgRNA1+sgHBGsense, and 55_sgRNA1+sgHBGsense. After gRNA1+sg1620 and 55_sgRNA1+sg1620+sgHBGsense are guided to the target site for site editing, the percentage of γ-globin transcribed mRNA in β-like globin mRNA (i.e. the sum of γ-globin and β-globin mRNA) will increase, with the percentages being 17.6% (Mock), 63.0% (55_sgRNA1), 39.3% (sg1620), 25.6% (sgHBGsense), 73.0% (sg1620+sgHBGsense), 84.8% (55_sgRNA1+sg1620), and 66.7% (55_sgRNA1+sg1620+sgHBGsense), respectively. It was demonstrated that ABE editing of the enhancer +55kb region of the BCL11A gene had a significant effect on enhancing HbF expression. When the enhancer +55kb region of the BCL11A gene was combined with the +58kb region or the enhancer +55kb region of the BCL11A gene was combined with the promoter region of the HBG1 / 2 genes, there was a more significant gain effect in enhancing HbF expression compared with editing of the +55kb single region.

[0309] According to the method in step 4 of implementation case 5, the cells that have completed in vitro differentiation are tested by HPLC. The results are as follows: Figure 19We used HPLC (DAD detector) to detect γ-globin levels in the ABE8e-55_sgRNA1 combination, which had high γ-globin expression levels as detected by qPCR. The results showed that compared with the unedited control mock, ABE8e editing at the 55_sgRNA1 site significantly increased γ-globin levels, with a γ-globin to α-globin ratio of 40.3%. γ-globin protein levels were significantly increased when ABE8e double-edited at the 55_sgRNA1 site and sg1620 site (60.8%), double-edited at the 55_sgRNA1 site and sgHBGsense site (48.5%), and co-edited at the 55_sgRNA1 site, sg1620 site, and sgHBGsense site (52.8%).

[0310] The editing effect in healthy humans showed that single editing of ABE8e at the 55_sgRNA1 site, double editing of ABE8e at the 55_sgRNA1 site and the sg1620 site, or double editing of ABE8e at the 55_sgRNA1 site and the sgHBGsense site significantly improved the mRNA and protein levels of γ-globin. The improvement effects were close to or exceeded the clinically reported cure threshold for SCD (HbF protein expression level reached 30%), suggesting that this editing strategy has great clinical application prospects in curing TDT or SCD. SEQUENCE LISTING <110> East China Normal University Shanghai Bangyao Biotechnology Co., Ltd. <120> A method, product and application for gene editing of single or multiple genes in cells <130> P21019765C <150> CN202011606753.9 <151> 2020-12-28 <160> 47 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgHBGsense <400> 1 cttgaccaat agccttgaca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgHBGsite1 <400> 2 cttgtcaagg ctattggtca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgHBGnspacer1 <400> 3 atgcaaatat ctgtctgaaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgHBGnspacer2 <400> 4 atatttgcat tgagatagtg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgHBGnspacer3 <400> 5 tatttgcatt gagatagtgt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgHBGnspacer4 <400> 6 atttgcattg agatagtgtg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgHBGnspacer5 <400> 7 gcattgagat agtgtgggga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sgHBGnspacer6 <400> 8 gtggggaagg ggcccccaag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sg1617 <400> 9 ctaacagttg cttttatcac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sg1618 <400> 10 ttgcttttat cacaggctcc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sg1619 <400> 11 ttttatcaca ggctccagga 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sg1620 <400> 12 tttatcacag gctccaggaa 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sg1621 <400> 13 cacaggctcc aggaagggtt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 55_sgRNA1 <400> 14 cactgatagg ggtcgcggta 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 55_sgRNA2 <400> 15 gcactgatag gggtcgcggt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2_Nontrans_sg1 <400> 16 acaagcatgg agcggctttg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2_Nontrans_sg2 <400> 17 cagctccatg gggcagaagt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2_Nontrans_sg3 <400> 18 ggccatgttc ctgcagttcc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2_Nontrans_sg4 <400> 19 cgggatggcc gctctgactc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2_Nontrans_sg5 <400> 20 ctccatggtg agcaacggta 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2_ABEskip_sg1 <400> 21 gcttaggcaa accagccttc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2_ABEskip_sg2 <400> 22 tccccaggtg accaatgctc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2_ABEskip_sg3 <400> 23 ctgcaggtgc acctgggaac 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2_ABEskip_sg4 <400> 24 caggatccgg cccctaccca 20 <210> 25 <211> 1610 <212> PRT <213> Artificial Sequence <220> <223> ABE8e <400> 25 His His His His His His Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu 1 5 10 15 Ser Pro Lys Lys Lys Arg Lys Val Ser Glu Val Glu Phe Ser His Glu 20 25 30 Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu 35 40 45 Arg Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile 50 55 60 Gly Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His 65 70 75 80 Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr 85 90 95 Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met 100 105 110 Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly 115 120 125 Val Arg Asn Ser Lys Arg Gly Ala Ala Gly Ser Leu Met Asn Val Leu 130 135 140 Asn Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu 145 150 155 160 Ala Asp Glu Cys Ala Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg 165 170 175 Gln Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly 180 185 190 Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu 195 200 205 Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys 210 215 220 Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala 225 230 235 240 Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu 245 250 255 Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu 260 265 270 Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr 275 280 285 Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln 290 295 300 Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His 305 310 315 320 Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg 325 330 335 His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys 340 345 350 Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp 355 360 365 Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys 370 375 380 Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser 385 390 395 400 Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu 405 410 415 Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile 420 425 430 Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala 435 440 445 Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala 450 455 460 Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala 465 470 475 480 Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu 485 490 495 Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu 500 505 510 Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg 515 520 525 Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys 530 535 540 Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val 545 550 555 560 Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser 565 570 575 Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu 580 585 590 Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu 595 600 605 Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg 610 615 620 Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu 625 630 635 640 His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp 645 650 655 Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr 660 665 670 Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg 675 680 685 Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp 690 695 700 Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp 705 710 715 720 Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr 725 730 735 Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr 740 745 750 Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala 755 760 765 Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln 770 775 780 Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu 785 790 795 800 Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His 805 810 815 Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu 820 825 830 Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu 835 840 845 Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe 850 855 860 Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp 865 870 875 880 Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser 885 890 895 Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg 900 905 910 Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp 915 920 925 Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His 930 935 940 Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln 945 950 955 960 Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys 965 970 975 Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln 980 985 990 Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly 995 1000 1005 Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu 1010 1015 1020 Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln 1025 1030 1035 Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 1040 1045 1050 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu 1055 1060 1065 Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys 1070 1075 1080 Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys 1085 1090 1095 Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile 1100 1105 1110 Thr Gln Arg Lys Phe Asp Asn With Thr Lys Ala Glu Arg Gly Gly 1115 1120 1125 Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val 1130 1135 1140 Glu Thr Arg Gln With Thr Lys His Val Ala Gln With Leu Asp Ser 1145 1150 1155 Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu With Arg Glu 1160 1165 1170 Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg 1175 1180 1185 Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His 1190 1195 1200 His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu 1205 1210 1215 Ile Lys Lys Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp 1220 1225 1230 Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln 1235 1240 1245 Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile 1250 1255 1260 Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile 1265 1270 1275 Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile 1280 1285 1290 Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu 1295 1300 1305 Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr 1310 1315 1320 Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp 1325 1330 1335 Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly 1340 1345 1350 Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala 1355 1360 1365 Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu 1370 1375 1380 Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn 1385 1390 1395 Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys 1400 1405 1410 Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu 1415 1420 1425 Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys 1430 1435 1440 Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr 1445 1450 1455 Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn 1460 1465 1470 Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp 1475 1480 1485 Glu Synthesis Glu Gln Synthesis Glu Phe Synthesis Lys Arg Val Synthesis 1490 1495 1500 Only Asp Only Asn Leu Asp Lys Val Leu Ser Only Tyr Asn Lys His 1505 1510 1515 Arg Asp Lys Pro With Arg Glu Gln Only Glu Asn With His Leu 1520 1525 1530 Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe 1535 1540 1545 Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val 1550 1555 1560 Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu 1565 1570 1575 Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser 1580 1585 1590 Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg 1595 1600 1605 Lys Val 1610 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Test115F <400> 26 tacaggcctc actggagcta 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Test115R <400> 27 gaagcgacct ggacttttgc 20 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Deepseq-HBG115-tryF <400> 28 ccccttcccc acactatctc a 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Deepseq-HBG115-tryR <400> 29 attcttcatc cctagccagc c 21 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 58-checksimilar-F <400> 30 agcatcacaa caggcagaga at 22 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 58-checksimilar-R <400> 31 gggaacacag atcctaacac agt 23 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> DeepSPCR-F-try <400> 32 gccagaaaag agatatggca tc 22 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DeepSPCR-R-try <400> 33 agagagcctt ccgaaagagg 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nontrans_PCR-F5 <400> 34 gggaactagg aggccaaagt 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nontrans_PCR-R2 <400> 35 tgggcatgag attggggcaa 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nontrans_PCR-F3 <400> 36 gtccgcccca aggtcaaaaa 20 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Nontrans_PCR-R5 <400> 37 aggcatctgg caataatgag gt 22 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2nontrans-DSF1 <400> 38 atcgctgggg gacagcctgc 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFR2nontrans-DSR2 <400> 39 ctcaggggct tgggaggggg 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HBG_-F <400> 40 tgggtcattt cacagaggag 20 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HBG_-R <400> 41 catcttccac attcaccttg c 21 <210> 42 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HBB_-F <400> 42 cagtgcaggc tgcctatc 18 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HBB_-R <400> 43 atacttgtgg gccagggcat 20 <210> 44 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HBA_-F <400> 44 gccctggaga ggatgttc 18 <210> 45 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HBA_-R <400> 45 ttcttgccgt ggccctta 18 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DHS+55-F2 <400> 46 ttcattggcc gaggatgacc 20 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DHS+55-R2 <400> 47 acctcactgg tttctcccct 20

Claims

1. A method for preparing a recombinant cell, characterized in that: The method includes the steps of introducing an adenine base editor and two sgRNAs into cells; Wherein, the two sgRNAs are an sgRNA targeting the sequence shown in SEQ ID NO: 14 and an sgRNA targeting the sequence shown in SEQ ID NO: 12; The adenine base editor comprises an amino acid sequence as shown in SEQ ID No: 25, or a sequence having more than 80% homology with the sequence shown in SEQ ID No:

25.

2. The method according to claim 1, wherein: The cells include hematopoietic stem / progenitor cells or erythroid progenitor cells; and / or, the cells are derived from mammals; and / or, the sgRNA includes chemical modifications of bases; And / or, the amino acid sequence of the adenine base editor is shown in SEQ ID No: 25; And / or, introducing the adenine base editor and at least one sgRNA into the cell includes using an electroporation method.

3. The method according to claim 2, wherein: The hematopoietic stem / progenitor cells include CD34+ hematopoietic stem / progenitor cells; And / or, the cells are derived from humans or mice.

4. A recombinant cell, characterized in that: The recombinant cell is prepared by the method according to any one of claims 1 to 3.

5. The recombinant cell according to claim 4, wherein The recombinant cells are hematopoietic stem / progenitor cells or erythroid progenitor cells; And / or, the recombinant cell is derived from a mammal.

6. The recombinant cell according to claim 5, wherein The recombinant cells are CD34+ hematopoietic stem / progenitor cells; And / or, the cells are derived from humans or mice.

7. A single-base gene editing system, characterized in that: The system comprises an adenine base editor as defined in any one of claims 1 to 3 and two sgRNAs as defined in any one of claims 1 to 3.

8. An sgRNA composition, comprising an sgRNA targeting the sequence shown in SEQ ID NO: 14 and an sgRNA targeting the sequence shown in SEQ ID NO:

12.

9. Use of the recombinant cell according to any one of claims 4 to 6, the single-base gene editing system according to claim 7, and / or the sgRNA composition according to claim 8 in preparing a gene editing product, a disease treatment and / or prevention product, or an animal model; Such diseases include beta-thalassemia and / or sickle cell anemia and / or dyserythropoiesis.

10. The use according to claim 9, characterized in that The cells targeted by the gene editing products include hematopoietic stem / progenitor cells or erythroid progenitor cells; And / or, the cells are derived from mammals.

11. The use according to claim 10, characterized in that The cells targeted by the gene editing products include CD34+ hematopoietic stem / progenitor cells; And / or, the cells are derived from humans or mice.

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