Strategy for precise editing of homologous regions

Abstract

Disclosed herein are methods for precise editing of highly homologous regions. The method includes identifying a target region for precise editing of a homologous region. A vector construct for expressing a fusion protein specific for the target region is obtained. The fusion protein is a genetic construct having sequences for expressing an inactivated Cas9 nuclease and an ABE8e base editor for editing at the identified target region. One or more vector constructs for expressing a guide RNA complementary to the target region are created. The gRNA vector construct(s) are transformed into a suitable cell line along with the fusion protein expression vector for precise editing of the highly homologous region. Also disclosed herein is another method for precise editing of highly homologous regions using a truncated guide RNA of 10-15 nucleotides in length for precise editing in highly homologous regions.

Description

Technical Field

[0001] This invention generally relates to genetic engineering for therapeutic purposes. More specifically, this invention relates to a method for precisely editing highly homologous regions without causing double-strand breaks. Background Technology

[0002] A series of mutations can occur in the coding regions of genes, leading to a variety of hereditary diseases. Some of these diseases can be treated using gene engineering techniques such as CRISPR-Cas9. However, because these techniques rely on DNA strand breaks, they can result in large deletions, especially in homologous regions, and in some cases, off-target editing. Therefore, there is an urgent need for an efficient gene editing method that can achieve high editing efficiency in the target region without creating cuts in the DNA, thereby preventing double-strand breaks or deletions of homologous regions.

[0003] In a known method, gene editing produces precise mutations without causing DNA double-strand breaks through base editing or amino acid substitution. Due to its versatility and ease of use, this technology is gaining increasing attention as a gene therapy tool. However, large fragment deletions occur when editing globin loci using base editors. Sequence homology between the A-γ and G-γ genes leads to deletions in the intercalary region between the two gRNA binding sites, accompanied by DNA nicks. The aforementioned method is inefficient and results in the deletion of homologous regions. Therefore, a new gene editing method is needed that can mediate base substitutions or mutations in homologous regions without causing DNA nicks and without resulting in homologous region deletions. Summary of the Invention

[0004] This invention provides a gene editing strategy to overcome hereditary diseases caused by gene mutations in gene coding regions with highly homologous regions.

[0005] One aspect of the present invention discloses a method for precisely editing highly homologous regions. The method includes: identifying a target region for precisely editing the homologous region; obtaining a vector construct for expressing a fusion protein specific to the target region, said fusion protein being a gene construct having a sequence for expressing an inactivated Cas9 nuclease (deadCas9) and an ABE8e base editor sequence for editing the identified target region; preparing one or more vector constructs for expressing a guide RNA complementary to the target region; and transforming the gRNA vector construct and the expression vector of the fusion protein together into a suitable cell line for precisely editing the highly homologous region.

[0006] Another aspect of the present invention relates to a method for precisely editing highly homologous regions. The method includes: obtaining one or more truncated guide RNAs (dead gRNA / dgRNA) targeting a specific region; obtaining one or more vector constructs for carrying a specially generated fusion complex for binding at the target region, each fusion complex comprising dgRNA, a Cas9 nickase, and a deaminase for binding at the target region; and transducing the generated vector constructs into a suitable cell line for precisely editing the homologous region.

[0007] The method disclosed in this paper can prevent double-strand breaks and off-target editing, and reduce insertion-deletion mutations (InDels) while generating point mutations. This method can avoid intercalary region deletions caused by cuts in homologous target regions. Attached Figure Description

[0008] To provide a detailed understanding of how the features of this invention are implemented, some embodiments of the invention are shown in the accompanying drawings. However, it should be noted that the accompanying drawings only illustrate typical embodiments of the invention and should not be considered as limiting the scope of the invention, as other equally effective embodiments are also possible.

[0009] Figure 1 According to one embodiment of the present invention, the deletion frequencies of gRNAs targeting different regions of the γ-globin promoter are shown.

[0010] Figure 1 b. According to one embodiment of the present invention, the deletion frequency of different base editors when using gRNA 2 is shown.

[0011] Figure 1 c. According to an example of the present invention, a comparison of the editing efficiency of base editors when using different gRNAs is shown.

[0012] Figure 1 According to one example of the present invention, the editing efficiency of gRNA 2, gRNA 3 and gRNA 11 is shown.

[0013] Figure 2 According to one example of the present invention, the deletion frequencies of different truncated gRNAs are shown when using the base editor nCas9.

[0014] Figure 2 b. According to an example of the present invention, the deletion frequency of different truncated gRNAs is shown when using the base editor dCas9.

[0015] Figure 2 c. According to an example of the present invention, the deletion frequency of different truncated gRNAs is shown when using the base editor ABE8e.

[0016] Figure 2 According to one example of the present invention, the deletion frequencies of different truncated gRNAs are shown when using the base editor dCas9 ABE8e.

[0017] Figure 3 According to another embodiment of the present invention, the editing efficiency of different truncated gRNAs targeting the target gene sequence is shown.

[0018] Figure 4 According to another embodiment of the present invention, the base editing efficiency of dead gRNA is shown.

[0019] Figure 5 According to another embodiment of the present invention, off-target editing of different truncated gRNAs at the HBD gene is shown.

[0020] Figure 6 According to one example of the present invention, gene editing at off-target sites is illustrated.

[0021] Figure 7 According to one embodiment of the present invention, the gene editing efficiency of nCas9ABE8e and dCas9ABE8e in the HBB and HBD regions is shown.

[0022] Figure 7 According to one embodiment of the present invention, a comparison is shown between unedited cells, cells transformed with nABE8e, and cells transformed with dABE8e to illustrate the fold change of the fusion protein and the absence of HBB and HBD regions.

[0023] Figure 7 According to one embodiment of the present invention, a comparison of the results of nCas9ABE8e and dCas9ABE8e is shown, wherein nCas9ABE8e produces bands corresponding to large fragment deletions.

[0024] Figure 7 According to one embodiment of the present invention, the editing efficiency of dABE8e and nABE8e before and after differentiation, as well as the identification of InDels, are shown.

[0025] Figure 7 E illustrates an increase in HbF-positive cells according to one embodiment of the present invention.

[0026] Figure 7 According to one embodiment of the present invention, a comparison of the frequencies of large fragment deletions caused by nABE8e and dABE8e, as measured by qRT-PCR, is shown.

[0027] Figure 7According to one embodiment of the present invention, a comparison of the frequencies of large fragment deletions caused by nABE8e and dABE8e, as determined by ddPCR, is shown.

[0028] Figure 7 According to one embodiment of the present invention, large fragment deletions caused by nABE8e and dABE8e are shown by PCR assessment.

[0029] Figure 8 According to one embodiment of the present invention, the percentage of nABE8e and dABE8e edited in CD90-positive cells is shown.

[0030] Figure 8 According to one embodiment of the present invention, the effect of increasing the base editor load on the editing percentage of nABE8e and dABE8e in CD90 positive cells is shown.

[0031] Figure 8 According to one embodiment of the present invention, the edit percentages of nABE8e and dABE8e in the HBB region are shown.

[0032] Figure 8 According to one embodiment of the present invention, the edit percentages of nABE8e and dABE8e in the HBG region are shown.

[0033] Figure 8 According to one embodiment of the present invention, the results of a clonogenic experiment were shown on samples including cells transformed with nABE8e, cells transformed with dABE8e, and unedited cells.

[0034] Figure 8 According to one embodiment of the present invention, the levels of DNA damage markers P21 and GADD45 in cells transformed with nABE8e and dABE8e are shown. Detailed Implementation

[0035] Unless otherwise stated, the definitions, terms and expressions used in this disclosure have their usual meanings and interpretations.

[0036] The term "Cas9" refers to a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNA that is complementary to the guide RNA.

[0037] The term "inactivated Cas9 (deadCas9 or dCas9)" refers to a mutant form of Cas9 whose endonuclease activity is eliminated by introducing a point mutation into its endonuclease domain.

[0038] The term "guide RNA (gRNA)" refers to a segment of RNA that acts as a guide to RNA- or DNA-targeting enzymes, forming a complex with the enzyme to remove, insert, or otherwise alter the targeted RNA or DNA.

[0039] The term "inactivated guide RNA (dgRNA)" refers to a truncated gRNA with a nucleotide length between 10 and 15 base pairs.

[0040] This invention provides a gene editing strategy that can prevent the deletion of homologous regions in gene sequences and double-strand breaks in gene sequences.

[0041] Various embodiments of the present invention provide a gene editing method. The method includes preparing inactivated Cas9 (dCas9). This protein can bind to a target region for gene modification without causing double-strand breaks in DNA. The obtained inactivated Cas9 is fused with a base editor and expressed as a fusion protein. In one example of the present invention, the base editor is an adenosine base editor, specifically ABE8e. The fusion protein is transformed into a suitable cell line for gene editing. In one example of the present invention, the cell line is mostly composed of cells carrying gene mutations, and the fusion protein is used to correct the gene mutation or edit the gene to improve various hereditary diseases. The transformed cells are overexpressed and validated to confirm successful gene editing for correcting the gene mutation. The method described herein will be described in detail below.

[0042] A Cas9 nuclease capable of catalytic inactivation that binds to a precise target region is generated; however, unlike the Cas9 nuclease itself, it does not induce double-strand breaks. In one example, Cas9 D10A is used, which cuts only the unedited DNA strand without inducing double-strand breaks. Subsequently, one or more base editors are screened to introduce point mutations in the target region. In one example, adenosine deaminase ABE8e is used as a base editor to generate point mutations. The screened base editor ABE8e is then fused with inactivated Cas9. This fusion protein efficiently performs base editing without inducing double-strand breaks and prevents the loss of homologous regions while preserving editing efficiency. Therefore, this method ensures a safe and reliable gene editing protocol. In one example, a fusion protein of dCas9 and ABE8e is obtained by introducing the H840A mutation into the HNH-like nuclease domain of the Cas9 nuclease. The obtained gene construct is transformed into the ABE8e vector to obtain a lentiviral construct. The construction of the fusion protein described herein will be described in detail below.

[0043] Vectors carrying the fusion protein are constructed by introducing the base editor sequence of adenosine deaminase into a suitable vector. The vectors are selected from a list including lentiviral vectors and adenovirus-associated virus (AAV) vectors. In one example, a lentiviral vector is used to construct the base editor construct. An ABE8e vector construct is obtained by inserting the ABE8e sequence into the lentiviral vector. In one example, the pLenti-ABE8e-puro vector is obtained by inserting the ABE8e sequence into the lentiviral vector. Similarly, a lentiviral vector carrying the fusion protein (dCas9 and ABE8e) is obtained by introducing an H840A mutation into the Cas9 domain of the pLenti-ABE8e-puro vector. Subsequently, multiple gRNAs complementary to the target region are designed. Each gRNA is transformed into a vector, thereby obtaining multiple vector constructs carrying that gRNA. Vectors used to construct the gRNA constructs are selected from a list including lentiviral vectors and u6 vectors.

[0044] In one example, gRNA was constructed in the pLKo5 vector to obtain gRNA vector constructs that are specific to the promoter regions of hemoglobin subunit γ1 and hemoglobin subunit γ2 (HBG1 and HBG2).

[0045] A vector construct carrying the inactivated Cas9 ABE8e gene sequence, along with multiple vector constructs for each gRNA, is transformed into suitable cells. These cells are cultured in suitable media to amplify the vector and overexpress it within the cell line. In one example, the pLenti-dCas9-ABE8e-puro vector is transformed into a suitable cell line. The cell lines are selected from, but are not limited to, HUDEP-2 cells, K562 cells, HEL cells, HEK cells, HL60 cells, and Jurkat cells. In one example, the obtained vector construct, along with a vector carrying the gRNA, is transformed into the HUDEP-2 cell line for target recognition and gene editing within the transformed cell line. The edited cells are cultured in suitable media and overexpressed. The resulting edited cells are analyzed to identify the correction of the gene mutation.

[0046] In one example, multiple gRNA constructs were obtained, selected from a set of guide RNAs consisting of sequence identification numbers gRNA2, gRNA3, and gRNA11, referring to the γ-globin loci G2, G3, and G11, respectively. These gRNAs were complementary to the promoter regions of HBG1 and HBG2. These gRNAs were first evaluated to determine the relationship between deletion frequencies and base editor activity. The guide RNA sequences used in this study are listed in the table below:

[0047] Table 1:

[0048]

[0049] Figure 1 According to one embodiment of the present invention, deletion frequencies among gRNAs targeting different regions of the γ-globin promoter are shown. An ABE7.10 base editor was used in conjunction with each guide RNA. gRNA 2, gRNA 3, and gRNA 11 were transduced into the HUDEP-2 cell line to assess the deletion frequencies of these three gRNAs. gRNA 3 (specific to the G3 locus) showed a lower degree of editing compared to gRNA 2 (specific to the G2 locus) and gRNA 11 (specific to the G11 locus). Figure 1 According to one embodiment of the present invention, the deletion frequencies of different base editors using gRNA 2 are shown. The efficiency of the adenosine base editor and the cytosine base editor was evaluated. The fold change in the 4.9 kb intergenic region compared with the locus control was evaluated when using the adenosine base editor (ABE7.10) and the cytosine base editor (CBE). It was observed that even when using the same gRNA, the deletion frequencies differed among the different base editors, and the editing efficiency was negatively correlated with the deletion frequency.

[0050] We further used gRNA 3 and gRNA 11 to determine the deletion frequencies of both ABE7.10 and ABE8e. Figure 1 According to one example of the present invention, a comparison of the editing efficiency of base editors when using different gRNAs is shown. Base editors ABE8e and ABE7.10 were used to compare the effects of the base editors relative to guide RNA sequences complementary to HBG1 and HBG2 (specifically gRNA3 and gRNA 11). gRNA 11 (G11) showed very little deletion in both base editors.

[0051] Based on the above research, each gRNA was further evaluated to assess the differences among different gRNAs in the 4.9kb deletion without considering the base editor. In one example, the gRNA was transduced as a lentivirus into D10A nickase HUDEP-2 stable cells without any fusion with the base editor to assess the genomic alteration and its functional consequences. Figure 1According to one example of the invention, the GC content of gRNA 2, gRNA 3, and gRNA 11 is shown. There is no correlation between the GC content assessment and the deletion frequency. The conversion efficiencies of gRNAs 2, 3, and 11 all exceed 90%. No base conversions or InDels were observed using the D10A cleavage enzyme Cas9, except for G11, which showed a small proportion of InDels, indicating that cleavage alone does not lead to the generation of InDels.

[0052] In one example, using the gene editing method described above, the study investigated the improvement of β-hemoglobinopathies by reactivating fetal hemoglobin expression to compensate for functional β-globin deficiency. The fusion protein inactivation of Cas9 and ABE8e, along with one or more guide RNAs specific to the HBG1 and HBG2 genes, was used for base editing. However, to validate this effective gene editing protocol and determine the frequency of large deletions in the promoter regions of the HBG1 and HBG2 genes, different base editors were used in this study. The promoter regions of the HBG1 and HBG2 genes were edited using dCas9, nCas9, ABE8e, and dCas9-ABE8e. Figure 2 a-2d, according to an example of the present invention, illustrates the missing frequencies when using base editors with different variants.

[0053] Figure 2 The planar plot (a) shows the fold change of various gRNAs in the 4.9kb intergenic region compared to the locus control when using the base editor nCas9 to determine deletion frequencies. Simple cuts are sufficient to produce large fragment deletions in homologous regions. Figure 2 The planar plot (b) shows the fold change of various gRNAs in the 4.9kb region between genes compared to the locus control when using the base editor dCas9 to determine deletion frequencies. Cells transformed with nuclease-deficient dCas9 showed the ability to prevent large deletions in homologous regions. Figure 2 The planar plot (c) shows the fold change of various gRNAs in the 4.9kb intergenic region compared to the locus control when using the base editor ABE8e, used to determine deletion frequencies. This study determined that the hyperactive variant ABE8e can improve editing efficiency and reduce large fragment deletions to a greater extent. Figure 2The planar plot (d) shows the fold change of various gRNAs in the 4.9kb intergenic region compared to the locus control when using the base editor dCas9-ABE8e, used to determine deletion frequencies. This study demonstrates that using the nuclease-deficient ABE8e (dCas9 ABE8e) can avoid large deletions and provide sufficient editing effects to boost fetal hemoglobin expression to therapeutically meaningful levels. The use of a nickase-deficient base editor variant was able to eliminate large deletions in the homologous γ-globin region.

[0054] In one example, the editing efficiency of the base editor dCas9-ABE8e in human CD34+ hematopoietic stem cells and progenitor cells (HSPCs) was evaluated. The aim of this study was to determine whether the base editor dCas9 ABE8e could prevent large deletions or intercalations in HSPCs during base editing of the γ-globin homologous region. First, the editing efficiency in CD34+ HSPCs was evaluated using a gRNA targeting exon 1 (with or without homology to the hemoglobin subunit δ (HBD) gene), which is of therapeutic significance, in the hemoglobin subunit β (HBB) gene. Cells were first nuclear transfected with nCas9ABE8e mRNA and dCas9 ABE8e mRNA, along with a guide RNA (sgRNA) complementary to the exon 1 region.

[0055] (Then) evaluate editing efficiency in the HBB and HBD regions. For example... Figure 7 As shown in diagram A, compared to cells targeted with nCas9ABE8e (nABE8e), the editing efficiency in both the HBB and HBD regions was slightly reduced when targeted with dCas9ABE8e (dABE8e). When using nABE8e, base transitions and large deletions at the HBD region were observed only in hemoglobin subunit β1 (HBB1) gRNA, which has binding sites at the hemoglobin subunit β (HBB) and hemoglobin subunit γ (HBG) genes, while this phenomenon was not observed only in hemoglobin subunit β2 (HBB2), which has a binding site at the HBB region. Samples in this study were obtained from healthy HSPC (hematopoietic stem cell) donors. Donors 1, 2, and 3 represent HSPCs obtained from healthy donor samples. The fold change of the fusion gene determines the deletion status in the homologous region. Figure 7 As shown in Figure B, real-time quantitative reverse transcription PCR (qRT PCR) using primers specific for large deletions between HBD and HBB showed that ABE8e exhibited a 10-fold increase in amplicons with large deletions compared to the unedited reference and the dABE8e-edited sample. Figure 7As shown in diagram C, large deletions were not detected even in cells exhibiting high editing efficiency at the HBB and HBG genes using HBB1-specific gRNA. Large deletions would result in smaller fusion genes that are detectable by PCR.

[0056] Further comparison of the nABE8e and dABE8e editors at the HBG promoter showed that the nABE8e base editor had an editing efficiency exceeding 90%. Editing was observed at the A4 and A5 base positions within the gRNA. However, as... Figure 7 As shown in planar plot D, dABE8e has slightly lower editing efficiency compared to ABE8e. By using AAVS1 incorporating both nABE8e and dABE8e, the increase in HbF-positive cells was identified in differentiated cells by comparing the percentage increase of control samples with that of unedited cells. Figure 7 As shown in diagram E, after differentiation, edited cells, whether integrated with gRNA 11 (ABE8e) or dABE8e, showed similar increases in HbF, reaching nearly 80%, indicating that the editing achieved by dABE8e was sufficient to increase HbF-positive cells with therapeutic significance. AAVS1 represents the sgRNA guiding the AAVS1 locus, while G11 represents the sgRNA guiding the γ-globin promoter. Furthermore, a large deletion was observed in cells edited with nABE8e, but not in cells edited with dABE8e. Figure 7 As shown in the planar graph F, a 4.9kb deletion was observed using the base editor ABE8e, as assessed by qRT-PCR. Figure 7 As shown in the planar diagram G, a comparison of 4.9kb deletion results in cells edited by ABE8e and nABE8e was evaluated by ddPCR. Figure 7 As shown in the planar diagram H, a 4.9kb deletion was observed in the sample edited with the base editor ABE8e by PCR evaluation, but no deletion was found in the unedited sample and dABE8e.

[0057] Furthermore, the activity of dABE8e in HSPCs was characterized to determine the extent of its editing efficiency and the reduction in InDel formation for therapeutic applications. It was observed that the editing efficiency of dABE8e was slightly lower than that of nABE8e in the homologous region. Further evaluation was conducted to determine whether the limited editing efficiency of dABE8e was more pronounced in pristine quiescent cells. The editing efficiency of nABE8e and dABE8e was compared in CD90-positive pristine HSPCs with that in bulk-edited cells. Figure 8As shown in the planar diagram A, base positions A4 and A5 did not show significant differences. Further analysis was then conducted to determine whether increasing the base editor load could improve editing efficiency in homologous regions. The results showed a moderate level of editing efficiency; however, as... Figure 8 As shown in Figure B, despite a fourfold increase in loading, the editing percentage did not improve, indicating that reagent availability was not a limiting factor for editing homologous regions.

[0058] The base editors nABE8e and dABE8e began editing within three hours of nuclear transfection; however, as... Figure 8 As shown in planar plots C (editing of the HBB region) and D (editing of the HBG region), the editing kinetics of nABE8e were significantly faster. This study performed colony formation experiments to determine the colony formation potential among nABE8e cell samples, dABE8e cell samples, and unedited cell (UE) samples. Colony formation experiments on edited cells showed no difference in colony formation potential among the samples, as... Figure 8 The planar diagram E is shown. In addition, the levels of DNA damage markers P21 and GADD45 were measured to determine the percentage of deletion. The levels of DNA damage response markers P21 and GADD45 in cells transfected with nABE8e and dABE8e nuclei were measured. Figure 8 As shown in F, nABE8e exhibited a slight increase in RNA levels, while dABE8e and the simulated electroporation sample showed similar levels. Therefore, dABE8e demonstrates a superior approach in terms of the purity of edited results, particularly for editing highly homologous regions and for multiple editing for therapeutic applications.

[0059] According to another embodiment of the present invention, a gene editing strategy is provided. This strategy relates to a method for base editing in homologous regions of a target gene sequence, capable of preventing large fragment deletions and reducing or preventing off-target base transitions. Furthermore, the present invention also prevents editing of unintended nucleotides. The gene editing strategy includes: obtaining one or more truncated guide RNAs (dead gRNAs) specific to the target region; subsequently obtaining a vector construct for carrying a fusion complex capable of binding at the target region. The vector for carrying the fusion complex is selected from a list including lentiviral vectors and adenovirus-associated virus (AAV) vectors. In one example, the vector construct is a lentiviral vector carrying a fusion complex comprising a truncated or dead gRNA construct, a Cas9 nickase, and a deaminase. The obtained vector construct is transformed into a suitable cell line. The cell lines are selected from, but are not limited to, HUDEP-2 cells, K562 cells, HEL cells, HEK cells, HL60 cells, and Jurkat cells. In one example, the obtained vector construct is transformed into HUDEP-2 cells for overexpression and evaluation of editing efficiency. The truncated gRNA was introduced into cells as a lentivirus for overexpression of the base editor. Editing efficiency was evaluated using Sanger sequencing after day 8.

[0060] In one example, the editing efficiency of truncated gRNAs of different lengths was evaluated. The truncated gRNAs used in this study included a dgRNA 2 with a length between 20 and 10 nucleotides. The 20-nucleotide dgRNA 2 sequence is designated by sequence ID dgRNA2.20 (G2.20), the 15-nucleotide dgRNA 2 sequence is designated by sequence ID dgRNA2.15 (G2.15), the 14-nucleotide dgRNA 2 sequence is designated by sequence ID dgRNA2.14 (G2.14), the 13-nucleotide dgRNA 2 sequence is designated by sequence ID dgRNA2.13 (G2.13), the 12-nucleotide dgRNA 2 sequence is designated by sequence ID dgRNA2.12 (G2.12), the 11-nucleotide dgRNA 2 sequence is designated by sequence ID dgRNA2.11 (G2.11), and the 10-nucleotide dgRNA 2 sequence is designated by sequence ID dgRNA2.10 (G2.10). The guide RNA sequences used in this study are listed in the table below:

[0061] Table 2:

[0062]

[0063] Figure 3According to one embodiment of the present invention, the editing efficiency of different truncated gRNAs targeting a gene sequence is illustrated. In this study, gRNA 2 with a length between 20 and 10 nucleotides was used. Guide RNA 2 (G2.20) with a length of 20 nucleotides was used as a control. gRNAs with a length of 14 nucleotides or less showed no significant editing effect, while gRNA with a length of 15 nucleotides (G2.15) showed an editing effect, but at a lower efficiency. A5, A8, A9, and A11 indicate the base positions within the gRNA where base editing may occur.

[0064] Further research was conducted on truncated gRNAs to evaluate their base editing efficiency in correcting HBE mutations in the β-globin gene. Figure 4 According to another embodiment of the present invention, the base editing efficiency of dead gRNA is shown. In this study, HBB guide RNA with sequence ID number HBB dsgRNA.20 and its truncated form were used. Its sequence is listed in Table 2. The 14-nucleotide-long HBB dsgRNA (HBB dsgRNA 14) was able to efficiently perform base editing at the target site; however, its editing efficiency was reduced by half compared to the 20-nucleotide-long gRNA (HBB dsgRNA 20).

[0065] Subsequently, the dead gRNA was evaluated to determine its editing efficiency in the HBD (hemoglobin subunit δ) and HBB (hemoglobin subunit β) regions. Aside from the presence of a single nucleotide mismatch, the HbE gRNA showed homology to the HBD gene. Many base editing protocols have resulted in deletions in the HBD / HBB intergenic region and caused off-target editing at the HBD. This gRNA also exhibited bystander editing at adjacent nucleotides resembling the expected nucleotide. In one example, the truncated gRNA did not show deletions at the HBD gene, indicating that the truncated gRNA is target-specific and does not cause off-target base switching. Figure 5 According to another embodiment of the present invention, off-target editing at the HBD gene using different truncated gRNAs is illustrated. In this study, the HBB guide RNA with sequence ID HBB dsgRNA.20 and its truncated form were used. Their sequences are listed in Table 2. Truncated gRNAs with 10 to 15 nucleotides did not exhibit editing at the HBD gene. This confirms the target specificity of the truncated gRNAs.

[0066] This study was further extended to determine gene editing in off-target regions at other genomic sites (such as the GDF6 gene on chromosome 8 and the SALC25A gene on chromosome 5), specifically using truncated gRNAs of lengths of 14 nucleotides, 15 nucleotides, and 20 nucleotides. Figure 6 Another example of the present invention illustrates gene editing at off-target sites. This analysis used HBB guide RNA with sequence ID HBB dsgRNA 20, and its truncated forms with sequence IDs HBB dsgRNA 14 and HBB dsgRNA 15. Their sequences are listed in Table 2. The analysis results show that when editing is performed using truncated gRNAs, off-target editing at other genomic sites (e.g., the GDF6 gene on chromosome 8, the SALC25A gene on chromosome 5, etc.) is significantly reduced. This provides an effective strategy for preventing off-target base switching.

[0067] This invention provides a method for achieving gene editing. This gene editing method prevents double-strand breaks in DNA. The method also ensures that large fragment deletions of homologous regions, off-target base switching, and bystander editing are prevented during gene editing. This invention provides an effective strategy by designing fusion proteins that inactivate Cas9-ABE8e, or by fusing deaminases (cytosine deaminase / adenosine deaminase) with other programmable nucleases whose nuclease domains are inactivated, in the form of DNA, mRNA, or protein, along with gRNA.

[0068] The present invention also provides another gene editing strategy, namely, using a fusion complex comprising truncated gRNAs (dead gRNA or dgRNA) of different lengths, in conjunction with a base editor in the form of DNA, mRNA or protein.

[0069] The fusion protein or fusion complex is delivered using a non-viral or viral base system. The delivery system includes, but is not limited to, electroporation, lentiviral vectors, AAV vectors, or lipid-based delivery methods, for altering the target gene sequence for gene therapy and other applications.

[0070] Target gene sequences for use in gene therapy and other applications.

[0071] The foregoing description of the present invention is for illustrative purposes only and is not intended to be limiting. Since modifications to the disclosed embodiments may occur to those skilled in the art within the scope and essence of the invention, the invention should be construed as covering everything within the scope of the appended claims and their equivalents.

Claims

1. A method for precise editing of highly homologous regions, the method comprising: Identify the target region; Create one or more carrier constructs that are complementary to the target region; Express the vector construct to obtain at least one of guide RNA, deadCas9 nickase, and ABE8e base editor; The expression vectors described herein were transformed into suitable cell lines to precisely edit the highly homologous regions of γ-globin; The method generates point mutations while preventing double-strand breaks, off-target editing, and reducing InDels.

2. The method according to claim 1, wherein, The target region is selected from at least one of the γ-globin loci of the HBG2 (HBD) gene and the HBG1 (HBB) gene.

3. The method according to claim 1, wherein, The vector constructs used to create the gene constructs are selected from a list including lentiviral vectors, adenovirus-associated virus vectors, and U6 vectors.

4. The method according to claim 1, wherein, The suitable cell line is at least one of HUDEP-2 cells, K562 cells, HEL cells, HEK cells, HL60 cells, and Jurkat cells.

5. The method according to claim 1, wherein, The highly homologous regions include, but are not limited to, the promoter regions of hemoglobin subunit γ1 (HBG1) and hemoglobin subunit γ2 (HBG2).

6. A method for precise editing of highly homologous regions, the method comprising: Identify the target region; Create one or more carrier constructs that are complementary to the target region; Express the vector construct to obtain at least one of a truncated guide RNA (dead gRNA), a Cas9 nickase, and a deaminase; The expressed vector construct was transformed into a suitable cell line to precisely edit the homologous region of γ-globin; The method generates point mutations while preventing double-strand breaks, off-target editing, and reducing InDels.

7. The method according to claim 6, wherein, The target region is selected from at least one of the γ-globin locus of the HBG2 (hemoglobin subunit δ / HBD) gene and the γ-globin locus of the HBG1 (hemoglobin subunit β / HBB) gene.

8. The method according to claim 6, wherein, The dgRNA is a truncated gRNA with base pairs ranging from 10 to 15 nucleotides.

9. The method according to claim 6, wherein, The vectors are selected from a list including lentiviral vectors and adenovirus-associated virus (AAV) vectors.

10. The method according to claim 6, wherein, The suitable cell line is at least one of HUDEP-2 cells, K562 cells, HEL cells, HEK cells, HL60 cells, and Jurkat cells.

11. The method according to claim 6, wherein, The highly homologous regions include, but are not limited to, the promoter regions of hemoglobin subunit γ1 (HBG1) and hemoglobin subunit γ2 (HBG2).

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