A method, composition and use for inducing high expression of delta-globin gene

Abstract

This invention belongs to the field of gene editing technology and discloses a method, composition, and application for inducing high expression of the δ-globin gene. The invention provides a method for inducing high expression of the δ-globin gene by artificially introducing a regulatory motif into the sense or antisense strand of the HBD gene regulatory region using gene editing technology; the regulatory motif is at least one of CCNCACCCT, NTG-N(7-8)-WGATAR, and CCAAT; or at least one of GGGTGNGG, YTATCW-N(7-8)-CAN, and ATTGG. This invention utilizes gene editing technology to edit the HBD gene to induce high expression of δ-globin. It eliminates the need for overexpression of exogenous globin genes, reducing the safety risks of gene therapy. Furthermore, it is applicable to various types of diseases caused by β-hemoglobin deletion or mutation, and is not limited to diseases caused by a single mutation site.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, specifically to a method, composition, and application for inducing high expression of the δ-globin gene. Background Technology

[0002] Hemoglobin is composed of globin and heme, and its main functions in the human body are carrying and transporting oxygen. In healthy adults, 95% of hemoglobin is HbA, which is composed of two α-globin chains and two β-globin chains; 2%-3.5% is HbA2, which is composed of two α-globin chains and two δ-globin chains.

[0003] β-thalassemia is a group of inherited hemolytic disorders primarily caused by gene mutations or deletions that reduce the synthesis of hemoglobin β chains. This leads to an imbalance in the synthesis ratio of α and β chains, resulting in α chain accumulation and ultimately hemolysis and other complications. Current treatments for thalassemia mainly involve regular blood transfusions and iron chelation therapy, but these methods still carry the risk of chronic damage to multiple organ functions. Hematopoietic stem cell transplantation is considered an effective cure for thalassemia, but most patients struggle to find suitable matches, and allogeneic transplantation carries varying degrees of graft-versus-host disease (GVHD). Furthermore, the high cost of transplantation hinders its widespread adoption.

[0004] In recent years, gene editing technology has made tremendous progress. The emergence of technologies such as CRISPR-Cas9, base editors, and leader editors has further broadened the application prospects of gene editing technology. Several clinical treatment protocols using CRISPR-Cas9 gene editing for β-thalassemia / sickle cell disease are underway and have achieved remarkable success. δ-globin, also a type of β-globin, combines with α-globin to form HbA2 (α2δ2), a ubiquitous and effective hemoglobin in adults, but at extremely low levels. Studies have shown that HbA2 has a lower affinity for oxygen and is ubiquitous in cells, while HbF is only present in 10%-30% of cells. Therefore, HbA2 is considered a more suitable replacement globin than HbF (α2γ2) for β-deficiency. δ-globin can balance the pathophysiological changes caused by the α-β imbalance resulting from β-globin deficiency, thus playing a role in treating β-thalassemia. Therefore, research on increasing δ-globin expression may become a new target for the treatment of β-thalassemia and SCD. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method, composition and application for inducing high expression of δ-globin gene.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for inducing high expression of the δ-globin gene by artificially introducing a regulatory motif into the sense or antisense strand of the HBD gene regulatory region using gene editing technology.

[0008] The artificially introduced sites include at least one of the following: upstream of the TSS of HBD, between -64 and -68, upstream between -89 and -81, upstream between -93 and -85, upstream between -100 and -92, upstream between -108 and -100, upstream between -119 and -111, upstream between -1470 and -1454, TSS between -78 and -62, downstream between +214 and +229, downstream between +1375 and +1391, and downstream between +1703 and +1719;

[0009] The regulatory motif is at least one of CCNCACCCT, NTG-N(7-8)-WGATAR, and CCAAT; or at least one of GGGTGNGG, YTATCW-N(7-8)-CAN, and ATTGG;

[0010] N is any one of A, T, G, and C; W is T or A; R is A or G; and Y is T or C.

[0011] This invention utilizes gene editing technology to edit the HBD gene to induce high expression of δ-globin. The method introduces CCNCACCCT, NTG-N(7-8)-WGATAR, and / or CCAAT motifs or their antisense complementary strands into the promoter region of the HBD gene, forming a positive regulatory element in the non-coding region of the HBD gene and promoting high expression of δ-globin in hematopoietic stem / progenitor cells.

[0012] As a preferred embodiment of the method described in this invention, the artificial introduction method includes at least one of sequence deletion, sequence insertion, and sequence mutation.

[0013] In a preferred embodiment of the method described in this invention, the regulatory motif is any one of CCAAT, CCACACCCT, TTATCAATTCTACTCAG, TTATCTTAAACCAACAG, CTGGGGTTTCTGATAG, CTATCTCTTCTCCGCAG, and CTGGCTCTACAGATAG.

[0014] As a preferred embodiment of the method described in this invention, the gene editing technology utilizes any one of the following editing systems: CRISPR-Cas editing system, base editor (BE), prime editing (PE), TALEN, ZFN, etc.

[0015] As a further preferred embodiment of the method described in this invention, the nucleotide sequence of the ssODN used by the CRISPR-Cas editing system includes at least one sequence as shown in SEQ ID No. 17 to SEQ ID No. 22, SEQ ID No. 50 to SEQ ID No. 54, or its corresponding antisense complementary sequence;

[0016] The nucleotide sequence of the sgRNA used includes at least one sequence as shown in SEQ ID No. 25 to SEQ ID No. 28, SEQ ID No. 56 to SEQ ID No. 60; and / or, the gRNA further comprises chemical modifications, preferably one or more of 3'-thiophosphate, 2'-O-methyl ester, 2'-O-methyl, 2'-F modification, 2'-ribose 3'-thiophosphate, deoxy, and 5' phosphate modification; more preferably, the three ribonucleotides at the 5' and 3' ends of the gRNA are modified with 2'-O-methyl, and the four ribonucleotides at the 5' and 3' ends are modified with thiophosphate bonds. In a second aspect, the present invention provides a composition for gene editing of the δ-globin gene regulatory region, comprising ssODN; wherein the ssODN contains at least one of CCNCACCCT, NTG-N(7-8)-WGATAR, CCAAT; or at least one of GGGTGNGG, YTATCW-N(7-8)-CAN, ATTGG;

[0017] N is any one of A, T, G, and C; W is T or A; R is A or G; and Y is T or C.

[0018] As a preferred embodiment of the composition of the present invention, the ssODN nucleotide sequence includes at least one sequence as shown in SEQ ID No. 17 to SEQ ID No. 22, SEQ ID No. 50 to SEQ ID No. 54, or its corresponding antisense complementary sequence.

[0019] (1) The CCNCACCCT, NTG-N(7-8)-WGATAR, CCAAT, GGGTGNGG, YTATCW-N(7-8)-CAN, and ATTGG sequences in the ssODN are located at any position on the ssODN, including the substitution sequence, the 5' homologous arm, the 3' homologous arm, the junction of the 5' homologous arm and the substitution sequence, or the junction of the 3' homologous arm and the substitution sequence.

[0020] (2) The 5' homologous arm and the 3' homologous arm are either symmetrical or asymmetrical;

[0021] (3) The 5' homologous arm and / or the 3' homologous arm are selected from the sense or antisense strand of the non-coding region of the ζ-globin gene;

[0022] (4) The 3' end of the 5' homologous arm in the ssODN is 0-20 bases away from the 5' end of the 3' homologous arm;

[0023] (5) The length of the 5′ homologous arm and / or the 3′ homologous arm is 20–300 nt; and,

[0024] (6) The number of bases in the substitution sequence is 0-6.

[0025] The ssODN is chemically modified, for example, by thiophosphate modification; preferably, the chemical modification is performed on the 5' and 3' ends of the ssODN; more preferably, the first three nucleotides of the 5' and 3' ends of the ssODN are modified.

[0026] As a further preferred embodiment of the composition described in this invention, the composition further includes sgRNA; the nucleotide sequence of the sgRNA includes at least one sequence as shown in SEQ ID No. 25 to SEQ ID No. 28, SEQ ID No. 56 to SEQ ID No. 60.

[0027] Thirdly, the present invention provides a reagent for gene editing of the regulatory region of the δ-globin gene, comprising the composition described above.

[0028] Fourthly, the present invention provides a hematopoietic stem / progenitor cell obtained by transferring the aforementioned composition into hematopoietic stem / progenitor cells.

[0029] Preferably, the hematopoietic stem / progenitor cells include CD34+ hematopoietic stem / progenitor cells;

[0030] Preferably, the cells are derived from mammals; more preferably, from humans or mice.

[0031] Fifthly, the present invention utilizes the described composition and the described hematopoietic stem / progenitor cells in the preparation of a medicament for treating diseases caused by β-hemoglobin deficiency or mutation. These diseases include β-thalassemia or sickle cell anemia.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] This invention introduces CCNCACCCT, CCAAT, or NTG-N(7-8)-WGATAR motifs into the non-coding region of the HBD gene, editing the bases within this region through insertion, deletion, or substitution to activate or enhance HBD expression. This eliminates the need for overexpression of exogenous globin genes, reducing the safety risks of gene therapy. Furthermore, activating δ-globin gene expression is applicable to various types of diseases caused by β-hemoglobin deficiency or mutation, and is not limited to diseases caused by a single mutation site. Attached Figure Description

[0034] Figure 1 This diagram illustrates the locations of the proposed CCAAT and CCNCACCCT library elements to be introduced into the upstream region of the HBD gene TSS, as well as the locations of the transcriptional elements of β-globin.

[0035] Figure 2 This is a schematic diagram showing the Sanger sequencing results of genes before and after electroporation editing at different sites in K562 cells.

[0036] Figure 3 Figure 3a shows the editing efficiency (3b) of introducing CCAAT and CCNCACCCT transcriptional elements into K562 cells and CD34+HSPC cells, as well as the results of qPCR detection and TOF detection (3c).

[0037] Figure 4 The NGS sequencing results for introducing the NTG-N(7-8)-WGATAR element into CD34+HSPC cells are shown in the figure.

[0038] Figure 5 Figure 5a shows the editing efficiency (5b) and TOF (5c) results of introducing NTG-N(7-8)-WGATAR transcriptional elements into K562 cells and CD34+HSPC cells. Detailed Implementation

[0039] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0040] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The non-coding region of the HBD gene includes 2kb upstream of the transcription start site (TSS), introns, and a 2kb range flanking the 3' region (Chromosome 11: 5232838-5236483; NCBI ID: NC_000011.10, genome version GRCh38.p14).

[0041] Example 1: Introducing binding sequences of NF-Y and KLF1 transcription factors into K562 cells

[0042] By introducing NF-Y and KLF1 binding sites into the upstream region of the HBD gene transcription start site (TSS), the specific locations of the candidate sites are as follows:

[0043] Region 1 (CCAAT), upstream of the TSS (promoter) of the HBD gene, between -64 and -68, the original sequence CCAAC;

[0044] Region 2 (TSS-81), between -89 and -81 upstream of the TSS of the HBD gene, original sequence TGAAACCCT;

[0045] Region 3 (TSS-85), between -93 and -85 upstream of the TSS of the HBD gene, original sequence CTAATGAAA;

[0046] Region 4 (TSS-92), between -100 and -92 upstream of the TSS of the HBD gene, contains the original sequence TCACAAACT;

[0047] Region 5 (TSS-100), between -108 and -100 upstream of the TSS of the HBD gene, contains the original sequence TTTCATTCT;

[0048] Region 6 (TSS-111), between -119 and -111 upstream of the HBD gene TSS, contains the original sequence GAAGGTTCA.

[0049] Based on the sequence characteristics of the candidate regions themselves and the cut site locations predicted by the CRISPR-Cas9 gene editing system, the most suitable mutation target types for each region were analyzed, as shown in Table 1. Underlined bases indicate the bases that need to be replaced, representing the desired result after gene editing. In this invention, the length of the replaced bases is 1-9 bases, achieving deletion, substitution, insertion, or a combination of these actions.

[0050] The six regions of the non-coding region of the δ-globin gene were altered by a few base substitutions, deletions, and insertions to form CCAAT or CCACACCCT and their antisense sequence structures. CCAAT is the binding sequence for NF-Y, and CCNCACCCT is the binding site for KLF1.

[0051] Table 1. Wild-type sequences and edited sequences of the HBD target editing region

[0052] CCAAT SEQ ID No.1 wild type TTATCTTAAACCAACCTGCTCACTG CCAAT SEQ ID No.2 Mutant 01 <![CDATA[TTATCTTAAA CCAAT CTGCTCACTG]]> TSS-81 SEQ ID No. 3 wild type CACAAACTAATGAAACCCTGCTTATCTTA TSS-81 SEQ ID No. 4 Mutant O2 <![CDATA[CACAAACTAA CC A C ACCCTGCTTATCTTA]]> TSS-85 SEQ ID No. 5 wild type TTCTCACAAACTAATGAAACCCTGCTTAT TSS-85 SEQ ID No. 6 Mutant 03 <![CDATA[TTCTCACAAA CC A CACCCT CCCTGCTTAT]]> TSS-92 SEQ ID No.7 wild type TTTTTCATTCTCACAAACTAATGAAACCC TSS-92 SEQ ID No. 8 Mutant 04 <![CDATA[TTTTTCATTC C CACA CC CTAATGAAACCC]]> TSS-100 SEQ ID No. 9 wild type AAGGTTCATTTTTCATTCTCACAAACTAA TSS-100 SEQ ID No. 10 Mutant 05 <![CDATA[AAGGTTCATT C CACA CC CTCACAAACTAA]]> TSS-111 SEQ ID No. 11 wild type ATAGTGGAATGAAGGTTCATTTTTCATTC TSS-111 SEQ ID No. 12 Mutant 06 <![CDATA[ATAGTGGAAT CC A CACC C T TTTTTCATTC]]>

[0053] To achieve the goal of cutting the DNA double strand near the editing site in the region selected in Table 1 to form a DSB, this invention selects the spCas9 CRISPR-Cas system (derived from Streptococcus pyogenes) or Cas12, which have high cutting efficiency, to analyze target sites with potentially high cutting efficiency. Sites 1 to 6 were selected as candidate target sites, and the DNA sequences and PAM sequences (NGG or NTTT) of the identified target sites are shown in Table 2 (SEQ ID No. 13 to SEQ ID No. 15).

[0054] Table 2 Target sites recognized by HBD non-coding region sgRNA / crRNA

[0055] SEQ ID No. 13 site_1 TCCTCCCTGCTCCAGTGAGC AGG CCAAT, TSS-81 antisense chain SEQ ID No. 14 site_2 TTTA AGATAAGCAGGGTTTCATTA TSS-85, TSS-92 antisense chain SEQ ID No. 15 site_3 TTTC ATTAGTTTGTGAGAATGAAA TSS-100 antisense chain SEQ ID No. 16 site_4 TTTG TGAGAATGAAAAATGAACCT TSS-111 antisense chain

[0056] Based on the expected mutation types shown in Table 1 and the DNA strands (sense or antisense strands) recognized by sgRNA and crRNA in Table 2, corresponding guide gene editing repair ssODNs were designed (Table 3, SEQ ID No. 17 to SEQ ID No. 22). In this invention, the ssODN structure includes a 5′ homologous arm, a substitution sequence, and a 3′ homologous arm. It should be noted that the CCAAT sequence, CCACACCCT sequence, or the corresponding antisense complement sequence in the ssODN structure can be located at any position on the ssODN, including the substitution sequence, the 5′ homologous arm, the 3′ homologous arm, and the junctions between the 5′ homologous arm and the substitution sequence, and between the 3′ homologous arm and the substitution sequence. The homologous arms on both sides of the ssODN can be symmetrical or asymmetrical (different lengths on both sides). For ease of systematic comparison, ssODNs with symmetrical homologous arms are used uniformly in this invention for experiments. The length of the homologous arms flanking the ssODN can range from 20 nt to 300 nt. For ease of systematic comparison, this embodiment uses an ssODN with a homologous arm length of approximately 60 nt. The ssODN can be the sense or antisense strand of the edited region DNA (the homologous arm sequence is the same as the corresponding sense strand or the antisense strand). In this invention, the DNA strand with the same recognition site as the sgRNA is uniformly selected as the ssODN master sequence. In addition to the homologous arm sequences flanking the ssODN, the substituted bases in the ssODN sequence can be 0, 1, 2, 3, 4, 5, or 6 bases, achieving deletion, substitution, insertion, or a combination of these. The effect can be changing more than one base on the genome, such as the deletion or substitution of 1-20 bases. As shown in Table 3, the underlined bases are the homologous arms flanking the ssODN, approximately 60 nt in length. The bases between the homologous arms that are not underlined are substituted bases. The first three nucleotides at both ends of ssODN are modified with phosphate thioate to enhance the stability of ssODN and improve its activity in gene editing.

[0057] Table 3 ssODN sequences and their applicable regions

[0058]

[0059]

[0060] Using the sgRNA corresponding to the target sites in Table 4, the first 20 bp is the sgRNA recognition site sequence, and the following 80 nt is the universal sgRNA backbone sequence SEQ ID No. 23: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU. The crRNA backbone sequence is SEQ ID No. 24: UAAUUUCUACUCUUGUAGAU.

[0061] Table 4 sgRNA sequences

[0062]

[0063] Using commercially available spCas9 and Cas12 proteins (purchased from Kaika Biotechnology, #Cas-EE109) ​​and chemically synthesized sgRNA (sequences shown in Table 4 for the corresponding sgRNA / crRNA, with chemically modified ends, 2'-O-methyl modification of the three ribonucleotides at the 5' and 3' ends, and phosphate thioester bond modification between the four ribonucleotides at the 5' and 3' ends), a ribonucleoprotein (RNP) complex was formed in vitro. The corresponding ssODN was added as a homology repair template strand. The RNP complex and ssODN were then delivered into K562 cells via electroporation. After culturing K562 cells for 48 hours following electroporation, genomic DNA was extracted from the cells, and the corresponding gene fragments were amplified by PCR and sequenced. The PCR primers for the corresponding sites are shown in Table 5. The Sanger sequencing base sequences before and after electroporation are shown in Table 5. Figure 2 The AF sequence results, analyzed using TIDE software, showed gene editing efficiency ( Figure 2 (In the K562 part), it can be seen that the editing efficiency of the selected 9 sites all reached more than 20%.

[0064] Table 5. PCR primer names and sequences for different sites

[0065]

[0066] K562 was further amplified and cultured for 7 days, and 1×10⁻⁶ samples were 6RNA was extracted from cells and reverse transcribed into cDNA. qPCR was performed using GAPDH as an internal control. The qPCR primers for the HBD gene were: forward primer 5'CAACCTCAAGGGCACTTTTT3' (SEQ ID No. 31) and reverse primer 5'AAGGGCACTTTTTCTCAGC 3' (SEQ ID No. 32). The GAPDH primers were: forward primer 5'CCATGGGGAAGGTGAAGGTC3' (SEQ ID No. 33) and reverse primer 5'GAAGGGGTCATTGATGGCAAC3' (SEQ ID No. 34). RT-qPCR results showed varying degrees of increase in δ-globin expression, with TSS-85 showing the most significant effect, achieving a 20-fold increase.

[0067] Example 2: Electroporation to introduce ssODN and spCas9 (or Cas12) protein / sgRNA RNP into human hematopoietic stem / progenitor cells to achieve efficient gene editing and enhance HBD expression.

[0068] After reviving human CD34-positive hematopoietic stem cells (HSPCs) mobilized from peripheral blood, they were placed in X-VIVO2+ enriched with human cytokines (SCF, TPO, Flt3L, 100 ng / mL each). TM After two days of culture in -15% serum-free Hematopoietic Cell Medium, the ssODN and spCas9 / sgRNA RNPs from Example 1 were delivered to HSPCs using the EO-100 program on a Lonza-4D electroporator. Following electroporation, the cells were cultured in X-VIVO2 enriched with human cytokines (SCF / TPO / Flt3L, 100 ng / mL each). TMAfter one day of recovery culture, the culture was then prepared with a solution containing EPO (24 IU / mL, PeproTech, catalog number: 100-64-250UG), SCF (100 ng / mL, Novoprotein, GMP-CD53), human AB serum (5%, GEMINI, catalog number: 100-512), insulin (10 μg / mL, Sigma, I2643-50MG), transferrin (330 μg / mL, Sigma, T4132-500MG), heparin (2 IU / mL, Sigma, H3149-500KU-9), and IL-3 (5 ng / mL, Sino...). Biology, catalog number: 11858-HNAE), hydrocortisone (1 μmol / L, manufacturer: Selleck, catalog number: S1696), glutamate (1%, manufacturer: Thermo, catalog number: 25030081), and other additives were cultured in IMDM medium. 2 × 10⁻⁶ samples were collected 48 hours after electroporation. 5 Genomic DNA was extracted from the cells, and the corresponding sequence fragments were amplified by PCR and then sequenced by Illumina (the sequencing primers for the corresponding sites are the same as those for K562 cells, as shown in Table 5). The sequencing results show that high editing efficiency can also be achieved in CD34+HSPC.

[0069] At HSPC-induced differentiation D14, take 1×10 6 RNA was extracted from cells and reverse transcribed into cDNA. qPCR was performed using GAPDH as an internal control (primers were the same as K562). RT-qPCR results showed an increase in δ-globin RNA levels, with TSS-85 showing the most significant effect, increasing levels by 3-4 times. Figure 2 b).

[0070] On day 21 of HSPC-induced differentiation, 2–4 × 10⁻⁴ samples were collected. 6 Cellular time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect the expression of different globins. Figure 2 C) The results showed that δ-globin levels were also increased in TSS-81, TSS-85, and TSS-92.

[0071] Example 3: Introducing the NTG-N(7-8)-WGATAR sequence into the HBD gene non-coding region of K562 cells and artificial hematopoietic stem cells.

[0072] Using gene editing technology, an NTG-N(7-8)-WGATAR or NAG-N(7-8)-WGATAR sequence is artificially formed in the sense or antisense strand of the non-coding region of the HBD gene. This causes the non-coding region of the HBD gene to form an enhancer element containing the NTG-N(7-8)-WGATAR sequence, the NAG-N(7-8)-WGATAR sequence, or the corresponding antisense complement sequence. The antisense complement sequence corresponding to the WGATAR sequence is YTATCW, where W is T or A, R is A or G, Y is T or C, and N is A, G, C, or T.

[0073] By analyzing the sequence within approximately 2 kb upstream of the HBD transcription start site (TSS), introns, and within 1 kb downstream of the 3' tail, the inventors discovered that many sequences could form NTG-N(7-8)-WGATAR or NAG-N(7-8)-WGATAR sequence structures by altering a few bases through substitution, deletion, or insertion. By analyzing whether the target mutant bases in these regions contained NGG (spCas9-recognized PAM) sequences and whether the cleavage site was precisely aligned with the target sequence, five candidate target regions most likely to achieve efficient gene editing were identified, listed in order of distance from the transcription start site:

[0074] Region 7 (T1400), upstream of the TSS (promoter) of the HBD gene, between -1470 and -1454, sequence TTATCAATTCTACTGTT;

[0075] Region 8 (T60), between -78 and -62 of the HBD gene promoter, sequence TTATTCTTAAACCAACCT;

[0076] Region 9 (N70) is located in the first intron of the HBD gene, between +214 and +229 downstream of the TSS, with the sequence CTTGGGTTTCTGATAG.

[0077] Region 10 (N800) is located in the second intron of the HBD gene, between +1375 and +1391 downstream of TSS, with the sequence CTACCTCTTCTCCGCAG.

[0078] Region 11 (W60) is located at the 3' end of the HBD gene, between +1703 and +1719 downstream of the TSS, with the sequence CTGAGGCTCTACAGATAG.

[0079] Based on the sequence characteristics of the five candidate regions and the cut site locations predicted by the CRISPR-Cas9 gene editing system, the most suitable mutation target types for each region were analyzed, as shown in Table 6. Underlined bases indicate bases that need to be replaced, representing the desired result after gene editing; "-" indicates deletion of that base. In this invention, the replacement base length is 0-6 bases, achieving deletion, replacement, insertion, or a combination of these actions.

[0080] The five regions of the non-coding region of the δ-globin gene were altered by a few base substitutions, deletions, and insertions to form the NTG-N(7-8)-WGATAR or NAG-N(7-8)-WGATAR sequence structures.

[0081] Table 6. Wild-type sequence of CTG-N(7-8)-WGATR to be introduced into the non-coding region of the HBD gene and the sequence obtained after editing.

[0082] T1400 SEQ ID No. 35 wild type CGTAATTTATCAATTCTACTGTTGGTAAG T1400 SEQ ID No. 36 Mutant 07 <![CDATA[CGTAATTTATCAATTCTACT CAG GGTAAG]]> T60 SEQ ID No. 37 wild type CCCTGCTTATCTTAAACCAACCTGCTCAC T60 SEQ ID No. 38 Mutant 08 <![CDATA[CCCTGCTTATCTTAAACCAA CAG GCTCAC]]> N70 SEQ ID No. 39 wild type AAGACTCTTGGGTTTCTGATAGGCACTG N70 SEQ ID No. 40 Mutant 09 <![CDATA[AAGACTCT G GGGTTTCTGATAGGCACTG]]> N800 SEQ ID No.41 wild type ATCTGCCTACCTCTTCTCCGCAGCTCTTG N800 SEQ ID No.42 Mutant 10 <![CDATA[ATCTGCCTA T CTCTTCTCCGCAGCTCTTG]]> W60 SEQ ID No. 43 wild type AGGTTCCTGAGGCTCTACAGATAGGGAGCA W60 SEQ ID No. 44 Mutant 11 AGGTTCCTG--GCTCTACAGATAGGGAGCA

[0083] Table 7 Target sites recognized by sgRNAs introducing CTG-N(7-8)-WGATR into the HBD untranslated region.

[0084] SEQ ID No. 45 site_5 TAATTTATCAATTCTACTGT TGG T1400 Chain of Justice SEQ ID No. 46 site_6 TCCTCCCTGCTCCAGTGAGC AGG T60 antisense chain SEQ ID No. 47 site_7 TGTAGACAGAGAAGACTCTT GGG N70 Chain of Justice SEQ ID No. 48 site_8 ACCTCTTCTCCGCAGCTCTT GGG NN800 Chain of Justice SEQ ID No. 49 site_9 GGTGTGTAAGAAGGTTCCTG AGG W60 Chain of Justice

[0085] Based on the expected mutation types shown in Table 6 and the DNA strands (sense or antisense strands) recognized by sgRNA in Table 7, corresponding guide gene editing repair ssODNs were designed (Table 8, SEQ ID No. 50 to SEQ ID No. 54). In this invention, the ssODN structure includes a 5′ homologous arm, a substitution sequence, and a 3′ homologous arm. It should be noted that the NTG-N(7-8)-WGATAR sequence, NAG-N(7-8)-WGATAR sequence, or the GATA or TATC sequence in the corresponding antisense complement sequence of the ssODN structure can be located at any position on the ssODN, including the substitution sequence, the 5′ homologous arm, the 3′ homologous arm, and the junctions between the 5′ homologous arm and the substitution sequence, and between the 3′ homologous arm and the substitution sequence. The homologous arms on both sides of the ssODN can be symmetrical or asymmetrical (different lengths on both sides). For ease of systematic comparison, this invention uniformly uses ssODNs with symmetrical homologous arms for experiments. The length of the homologous arms flanking the ssODN can range from 20 nt to 300 nt. For ease of system comparison, this embodiment uses an ssODN with a homologous arm length of approximately 60 nt as an example. The ssODN can be the sense or antisense strand of the edited region DNA (the homologous arm sequence is the same as the corresponding sense strand or the antisense strand). In this invention, the DNA strand with the same recognition site as the sgRNA is uniformly selected as the ssODN master sequence. In addition to the homologous arm sequences flanking the ssODN, the substituted bases in the ssODN sequence can be 0, 1, 2, 3, 4, 5, or 6 bases, achieving deletion, substitution, insertion, or a combination of deletion, substitution, and insertion. The achieved effect can be a change of more than one base on the genome, such as the deletion or substitution of 1-20 bases. As shown in Table 8, the underlined bases are the homologous arms flanking the ssODN, with a length of approximately 60 nt. The bases between the homologous arms that are not underlined are the substituted bases. The first three nucleotides at both ends of ssODN are modified with phosphate thioate to enhance the stability of ssODN and improve its activity in gene editing.

[0086] Table 8 ssODN sequences and their applicable regions

[0087]

[0088] Backbone sequence SEQ ID No. 55:

[0089] GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU.

[0090] Table 9 sgRNA Sequences

[0091]

[0092] Using commercially available spCas9 protein (purchased from Kaika Biotechnology, #Cas-EE109) ​​and chemically synthesized sgRNA (sequences shown in Table 9, with chemically modified ends, 2'-O-methyl modification of the three ribonucleotides at the 5' and 3' ends, and phosphate thioester bond modification between the four ribonucleotides at the 5' and 3' ends), a ribonucleoprotein (RNP) complex was formed in vitro. The corresponding ssODN was added as a homology repair template strand. The RNP complex and ssODN were then delivered into K562 / CD34+HSPCs via electroporation. After 48 hours of cell culture following electroporation, genomic DNA was extracted from the cells, and the corresponding gene fragments were amplified by PCR and sequenced. The PCR primers for the corresponding sites are shown in Table 10. The NGS deep sequencing results of the edited sites are shown in Table 10. Figure 4 and Figure 5 a. K562 cells were cultured for another 7 days. A subset of CD34+ HSPCs were then differentiated, and the relative expression levels of RNA were measured on day 14. Using RT-qPCR with GAPDH as an internal control, the results showed that the T60 site among the five candidate sites exhibited a more than 30-fold activation effect in K562 cells, while the RNA level of δ-globin in CD34+ HSPC cells was 3-4 times that of the control group. MALDI-TOF MS analysis of CD34+ HSPC cells on day 21 of differentiation showed that the protein level at this site was also increased.

[0093] Table 10. PCR primer names and sequences for different sites

[0094] SEQ ID No. 61 HBD-T1400-F TAGTTTGAACTCACCTCTGG TSS1400 SEQ ID No. 62 HBD-T1400-R ACACATGCATGTGTGTATCC TSS1400 SEQ ID No. 63 HBD-sanger-F ATCTCTAGAGGCAAAGAAGAACTTT T60 SEQ ID No. 64 HBD-sanger-R AGGGCATTGACAGCAGTCTT T60 SEQ ID No. 65 HBD-N70-F CAAAGTGAACGTGGATGCAG N70 SEQ ID No. 66 HBD-N70-R CTTAGGGTTGCCCATAACAG N70 SEQ ID No. 67 HBD-N800-F GGGAATAACCTGGGGATCAG N800 SEQ ID No. 68 HBD-N800-R CAGCCACCACCTTCTGATAG N800 SEQ ID No. 69 HBD-W60-F AGAATGTTCAGCTCAACTTCC W60 SEQ ID No. 70 HBD-W60--R GCCTTGTACGGTTCCCTTG W60

[0095] This invention significantly enhances the expression of the δ-globin gene and protein after gene-edited cells differentiate into erythrocytes by forming CCAAT, CCNCACCCT, and NTG-N(7-8)-WGATAR as enhancer elements in different regions of the δ-globin gene. Therefore, this invention has potential application value in gene therapy for diseases caused by β-hemoglobin deficiency or mutation (such as β-thalassemia or sickle cell anemia).

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for inducing high expression of δ-globin gene for non-therapeutic and diagnostic purposes, characterized by, Regulatory motifs were artificially introduced into the antisense strand of the non-coding region of the HBD gene using gene editing technology. The artificially introduced site is between -78 and -62 of the HBD gene promoter, and the sequence before the artificially introduced regulatory motif is TTATTCTTAAACCAACCT; The sequence after artificially introducing the regulatory motif is TTATCTTAAACCAACAG; The non-coding region of the HBD gene includes a 2kb range upstream of the transcription start site, an intron, and a 2kb range flanking the 3′ region; Chromosome 11: 5232838-5236483; NCBI number NC_000011.10, genome version GRCh38.p14; The gene editing technology utilizes the CRISPR-Cas editing system.

2. The method according to claim 1, characterized in that, The nucleotide sequence of ssODN used in the CRISPR-Cas editing system is shown in SEQ ID No. 51; The nucleotide sequence of the sgRNA used is shown in SEQ ID No. 57.

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