WO2021121321A1 - 一种提高基因编辑效率的融合蛋白及其应用 - Google Patents
一种提高基因编辑效率的融合蛋白及其应用 Download PDFInfo
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- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
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Definitions
- the invention relates to the field of biotechnology, in particular to a fusion protein for improving gene editing efficiency and its application.
- CRISPR/Cas9 derived from Streptococcus pyogenes spCas9 to use NGG as the PAM (spacer sequence precursor adjacent motif) and recognizes and specifically binds to the single base from C to T or G to A upstream of NGG. Base mutation.
- BE3 base editor 3
- UGI uracil glycosidase inhibitor
- ⁇ -hemoglobinopathy such as ⁇ -thalassemia and sickle cell disease (SCD) is caused by mutations in the HBB gene encoding ⁇ -hemoglobin.
- SCD sickle cell disease
- HPFH hereditary fetal hemoglobinopathy
- HPFH hereditary fetal hemoglobinopathy
- the high expression of gamma-globin in adult patients can alleviate the disease phenotype caused by beta hemoglobin mutations. It has been reported that deleting 13bp of the promoter region of HBG1/2 with CRISPR/Cas9 can activate the expression of ⁇ -globulin, thereby alleviating or treating thalassemia disease, which is an effective treatment strategy.
- a heterozygous point mutation at position -117 in the HBG1/2 promoter region produced 10-20% of fetal hemoglobin (HbF) expression.
- the mechanism is -117G>A
- the mutation destroys the binding site of the transcription repressor BCL11A.
- the purpose of the present invention is to provide a fusion protein that improves gene editing efficiency and its application.
- the present invention provides a fusion protein that improves gene editing efficiency, including a single-stranded DNA binding protein functional domain, nucleoside deaminase and nuclease.
- connection sequence of the fusion protein is: the nucleoside deaminase is located at the N-terminus or the C-terminus of the nuclease, and the single-stranded DNA binding protein functional domain is located at the nucleoside deaminase and the nucleoside deaminase.
- the nucleoside deaminase is located at the N-terminus of the nuclease
- the single-stranded DNA binding protein functional domain is located between the nucleoside deaminase and the nuclease.
- the single-stranded DNA binding protein includes a sequence-specific single-stranded DNA binding protein, and/or a non-sequence-specific single-stranded DNA binding protein, preferably, a non-sequence-specific single-stranded DNA binding protein,
- the non-sequence-specific single-stranded DNA binding protein is selected from RPA70 (subunit 70 of human replication protein A), RPA32 (subunit 32 of human replication protein A), BRCA2 (breast cancer No. 2 gene), hnRNPK (Heterogeneous nuclear ribonucleoprotein K), PUF60 (poly-U binding splicing factor 60KDa) and Rad51 (a homologous recombination repair protein) any one or more;
- sequence-specific single-stranded DNA binding protein is selected from any one of TEBP (telomere binding protein), Teb1 (a constituent protein of telomerase) and POT1 (human telomere protective protein 1) or Any number
- the single-stranded DNA-binding protein functional domain includes at least one of the following four domains (any one, any two, any three or all four) or the following four domains have a single Partial polypeptide fragments with chain DNA binding function and any combination thereof: OB folding (oligonucleotide/oligosaccharide/oligopeptide binding folding), KH domain (K homology domain), RRMS of the single-stranded DNA binding protein (RNA recognition motif), whirly domains;
- the single-stranded DNA binding protein functional domain includes the DNA binding domain (DBD) of Rad51. More preferably, the amino acid sequence of the DNA binding domain of Rad51 includes the sequence shown in SEQ ID No. 1, and more Preferably, the coding sequence of the DNA binding domain of Rad51 includes the sequence shown in SEQ ID No. 2;
- the amino acid sequence of the DNA binding domain of RPA70 includes the sequence shown in SEQ ID No. 11, and more preferably, the coding sequence of the DNA binding domain of RPA70 includes the sequence shown in SEQ ID No. 12.
- the deaminase includes cytosine deaminase (APOBEC) and/or adenosine deaminase, preferably, cytosine deaminase, and the cytosine deaminase can be derived from different organisms. body,
- the cytosine deaminase includes rat-derived cytosine deaminase.
- the rat-derived cytosine deaminase amino acid sequence includes SEQ ID No. 3
- the sequence, more preferably, the coding sequence of the rat-derived cytosine deaminase includes the sequence shown in SEQ ID No. 4;
- the cytosine deaminase includes human-derived cytosine deaminase APOBEC3A.
- the amino acid sequence of the human-derived cytosine deaminase APOBEC3A includes SEQ ID No. 13 More preferably, the coding sequence of the cytosine deaminase APOBEC3A includes the sequence shown in SEQ ID No. 14;
- the cytosine deaminase includes a mutant of cytosine deaminase APOBEC3A, and the cytosine deaminase APOBEC3A mutant is the cytosine deaminase APOBEC3A at position 57 (from the start Codon) asparagine (N or Asn) is mutated to glycine (G or Gly), preferably, the cytosine deaminase APOBEC3A is derived from humans, more preferably, the amino acid sequence of the cytosine deaminase APOBEC3A includes The sequence shown in SEQ ID No.
- the coding sequence of the cytosine deaminase APOBEC3A includes the sequence shown in SEQ ID No. 14; the amino acid sequence of the cytosine deaminase APOBEC3A mutant includes SEQ ID No.
- the sequence shown in .15, more preferably, the coding sequence of the cytosine deaminase APOBEC3A includes the sequence shown in SEQ ID No. 16;
- the nuclease is selected from one or more of Cas9, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, Cpf1; preferably
- the nuclease is Cas9; more preferably, the Cas9 is selected from Cas9 derived from Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes or Streptococcus thermophilus, more preferably, the Cas9 is selected from Cas9 Mutants VQR-spCas9, VRER-spCas9, spCas9n, more preferably, spCas9n, more preferably, the amino acid sequence of spCas9n includes the sequence shown in SEQ ID No. 5, and more preferably, the coding sequence of spCas9n includes
- NLS Nuclear Localization Signal
- the NLS is located at at least one end (C-terminal and/or N-terminal) of the fusion protein; more preferably, the amino acid sequence of the NLS includes The sequence shown in SEQ ID No. 7, and more preferably, the coding sequence of the NLS includes the sequence shown in SEQ ID No. 8;
- the fusion protein also includes UGI (uracil glycosidase inhibitor).
- UGI uracil glycosidase inhibitor
- the UGI is located at at least one end (C-terminal and/or N-terminal) of the fusion protein; more preferably, the amino acid sequence of the UGI It includes the sequence shown in SEQ ID No. 9, and more preferably, the coding sequence of the UGI includes the sequence shown in SEQ ID No. 10, and more preferably, the UGI is more than two copies.
- the present invention also provides any one of the following A)-C) biological materials:
- a gene encoding any one of the above-mentioned fusion proteins is DNA or RNA (such as mRNA);
- Immune cells such as T cells
- hematopoietic stem cells such as T cells
- bone marrow cells such as red blood cells, preferably red blood cell precursor cells or hematopoietic stem cells.
- the present invention also provides a sgRNA for gene-editing a target gene in a cell, and the target sequence of the sgRNA includes at least one of SEQ ID No. 17-36,
- the cells are T cells, hematopoietic stem cells, bone marrow cells or red blood cells, more preferably, red blood cell precursor cells or hematopoietic stem cells,
- the target genes are HBG1 and HBG2 promoter regions (specifically, the G at position -117 is edited as A, and the G at position -117 is the 14th G from the left in the promoter region CCAGCCTTGCCTTGACCAATAGCC).
- the present invention also provides a single-base gene editing system, comprising any one of the above fusion protein or the biological material and sgRNA, and the sgRNA guides the purpose of the fusion protein to the target cell.
- Single-base gene editing of genes comprising any one of the above fusion protein or the biological material and sgRNA, and the sgRNA guides the purpose of the fusion protein to the target cell.
- the target sequence of the sgRNA includes at least one of SEQ ID No. 17-36;
- the cells are T cells, hematopoietic stem cells, bone marrow cells, red blood cells, or red blood cell precursor cells,
- the target genes are HBG1 and HBG2 promoter regions.
- the present invention protects any of the fusion proteins, the biological materials, and the single-base gene editing system in the preparation of gene editing products, disease treatment and/prevention products, animal models, or new plant varieties.
- the disease is beta hemoglobinopathy
- the beta hemoglobinopathy includes beta thalassemia and/or sickle cell anemia.
- the present invention also provides a method for improving the efficiency of single-base gene editing, including the steps of introducing any one of the above fusion proteins and sgRNA into cells and performing gene editing on the target gene.
- the sgRNA guides the The fusion protein performs single-base gene editing on the target gene.
- the target sequence of the sgRNA includes at least one of SEQ ID No. 17-36;
- the cells are T cells, hematopoietic stem cells, bone marrow cells, red blood cells, or red blood cell precursor cells;
- the target genes are HBG1 and HBG2 promoter regions.
- the present invention also provides a method for constructing an animal model of a disease, including the steps of introducing any one of the above-mentioned fusion proteins and sgRNA into animal cells and performing gene editing on the target gene;
- the target sequence of the sgRNA includes at least one of SEQ ID No. 17-36; more preferably, the target sequence of the sgRNA includes the sequence shown in SEQ ID No. 36, and the target gene includes the DMD gene ;
- the animal is a mammal, more preferably, the mammal is a rat or a mouse, more preferably a mouse;
- the cell is an embryonic cell
- the method of introduction is one of vector transformation, microinjection, transfection, lipofection, heat shock, electroporation, transduction, gene gun, DEAE-dextran-mediated transfer, or Any combination, more preferably, microinjection;
- the introduction is carried out using the mRNA of any one of the above-mentioned fusion proteins and the sgRNA,
- the concentration of the mRNA of any one of the above fusion proteins used in the introduction is 1-1000ng/ ⁇ L, more preferably, 10-600ng/ ⁇ L, more preferably, 50-150ng/ ⁇ L, more preferably, 100ng / ⁇ L
- the concentration of the introduced sgRNA used is 1-1000ng/ ⁇ L, more preferably, 10-600ng/ ⁇ L, more preferably, 150-250ng/ ⁇ L, more preferably, 200ng/ ⁇ L
- the concentration ratio of the mRNA of any of the above fusion proteins used in the introduction to the sgRNA used in the introduction is 1:(5-1), more preferably, 1:(4-1.5), More preferably, 1:(3-1.8), more preferably, 1:2.
- the invention protects the application of the animal model obtained by the method in drug screening, disease treatment effect evaluation or disease treatment mechanism research.
- the present invention provides a product for the treatment and/or prevention of ⁇ -hemoglobinopathy, comprising: a delivery vector of the gene and sgRNA described in A) above,
- the sgRNA guides the fusion protein to affect the HBG1 and HBG2 promoter regions in the target cell (specifically, the G at position -117 is edited as A, and the G at position -117 is the 14th position from the left in the promoter region CCAGCCTTGCCTTGACCAATAGCC G) Perform single-base gene editing;
- the target sequence of the sgRNA includes the sequence shown in SEQ ID No. 35;
- the beta hemoglobinopathy includes beta thalassemia and/or sickle cell anemia
- the cells are T cells, hematopoietic stem cells, bone marrow cells or red blood cells, more preferably, red blood cell precursor cells or hematopoietic stem cells,
- the target genes are HBG1 and HBG2 promoter regions (specifically, the G at position -117 is edited as A, and the G at position -117 is the 14th G from the left in the promoter region CCAGCCTTGCCTTGACCAATAGCC).
- the delivery vector includes a viral vector and/or a non-viral vector;
- the viral vector includes an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, a retroviral vector, and/or an oncolytic viral vector ,
- the non-viral vector includes cationic polymer, plasmid vector and/or liposome;
- the delivery vehicle includes a lentiviral vector.
- the homology with the sequence described in this application is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or More than 99% of the sequence, and/or the sequence after substitution, deletion or insertion of amino acid residues or nucleotides based on the sequence described in this application, and a sequence with the same or similar functions as the sequence used in this application , Are all within the scope of protection of this application.
- the present invention uses CBEs (pyrimidine base conversion technology) in the process of CG to TA base conversion.
- Nucleoside deaminase such as cytosine deaminase, uses single-stranded DNA as a substrate for deamination.
- the single-stranded DNA binding protein functional domain is fused to the nuclease fusion protein, which greatly increases the chance of single-stranded DNA being exposed to nucleoside deaminase, thereby significantly improving the base editing efficiency.
- the present invention screened the functional domains of 10 non-sequence-preferred single-stranded DNA binding proteins to be fused with BE4max, and found that a single-stranded DNA binding functional domain (1-114AA) derived from human Rad51 was fused in the middle of Apobec1 and Cas9n. The highest efficiency improvement was produced, named hyBE4max. Compared with BE4max, the C-G to T-A editing efficiency of hyBE4max is increased by 16 times, especially for sites close to the PAM region, while maintaining low indels (insertions or deletions).
- the present invention has made a breakthrough improvement on single-base gene editing technology, and can greatly promote its application in gene editing, gene therapy, cell therapy, animal model making, crop genetic breeding and the like.
- the present invention takes ⁇ -hemoglobinopathy as an example.
- A3A-BE4max, hyeA3A-BE4max targets HBG1 and HBG2 (hereinafter referred to as HBG1/2) promoter regions closer to PAM region -117 It can more accurately and efficiently target -117 to generate G to A mutations, thereby activating the expression of ⁇ -globin, providing a more precise and efficient treatment strategy for clinical treatment of ⁇ -hemoglobinopathy.
- HBG1/2 HBG1 and HBG2
- the present invention applies hyA3A-BE4max to the production of mouse disease animal models. Compared with A3A-BE4max, hyA3A-BE4max targets C to T mutations near the PAM region to produce disease animal models. Therefore, the present invention provides a A new type of platform for efficiently producing disease animal models will greatly promote the production process of different animal models.
- FIG. 1 is a schematic diagram of the fusion of different single-stranded DNA binding protein functional domains with BE4max.
- NLS is a nuclear localization signal (its amino acid sequence is shown in SEQ ID No. 7, and its coding sequence is shown in SEQ ID No. 8), and rA1 is cytidine deaminase APOBEC1 (its amino acid sequence is shown in SEQ ID No. 3).
- the coding sequence is shown in SEQ ID No. 4
- spCas9n is Cas9n derived from Streptococcus pyogenes (its amino acid sequence is shown in SEQ ID No. 5, and the coding sequence is shown in SEQ ID No.
- UGI is a uracil glycosidase inhibitor (its amino acid sequence is shown in SEQ ID No. 9 and the coding sequence is shown in SEQ ID No. 10), and SSDBD is a single-stranded DNA binding protein functional domain.
- Figure 2 shows the comparison of C to T base editing efficiency (ie, the ordinate, in %) achieved by hyBE4max and BE4max on 8 targets on 293T.
- Figure 3 is a comparison of the average C to T base editing efficiency (ie, the ordinate, in %) produced by hyBE4max and BE4max on 8 targets on 293T.
- Figure 4 is a comparison of the base editing efficiency (ie, the ordinate, the unit is %) of indels generated by hyBE4max and BE4max on 8 targets on 293T.
- FIG. 5 is a schematic diagram of the structure of the fusion proteins A3A-BE4max and hyA3A-BE4max.
- hA3A is a human-derived cytidine deaminase APOBEC3A (its amino acid sequence is shown in SEQ ID No. 13, and the coding sequence is shown in SEQ ID No. 14), and NLS, spCas9n and UGI are the same as in Fig. 1.
- Figure 6 is a comparison of the C to T base editing efficiency (ie, the ordinate, in %) achieved by hyA3A-BE4max and A3A-BE4max on 8 endogenous targets on 293T.
- Fig. 7 is a comparison of the average C to T base editing efficiency (i.e. the ordinate, the unit is %) achieved by hyA3A-BE4max and A3A-BE4max on 8 endogenous targets on 293T.
- Fig. 8 is a comparison of the base editing efficiency (ie, the ordinate, the unit is %) of indels generated by 8 endogenous targets on 293T between hyA3A-BE4max and A3A-BE4max.
- Figure 9 is a schematic diagram of the structure of the fusion proteins eA3A-BE4max and hyeA3A-BE4max.
- A3A N57G is the N57G mutant of hA3A used in Figure 5 (its amino acid sequence is shown in SEQ ID No. 15, and the coding sequence is shown in SEQ ID No. 16), and NLS, spCas9n and UGI are the same as in Figure 1.
- Figure 10 is a comparison of the C to T base editing efficiency (i.e. the ordinate, the unit is %) achieved by hyeA3A-BE4max and eA3A-BE4max on 11 endogenous targets on 293T.
- Figure 11 is a comparison of the average C to T base editing efficiency (i.e. the ordinate, the unit is %) achieved by hyeA3A-BE4max and eA3A-BE4max on 11 endogenous targets on 293T.
- Figure 12 is a comparison of the base editing efficiency (ie the ordinate, the unit is %) of indels generated by 11 endogenous targets on 293T between hyeA3A-BE4max and eA3A-BE4max.
- the abscissa C in Figures 2, 3, 5, 6, and 11 represents the position of the C edited as T on the corresponding target sequence.
- C5 represents the position from the 5'end of the corresponding target sequence.
- the 5-digit C is edited as the efficiency of T.
- Figure 13 is a schematic diagram of hyeA3A-BE4max targeting HBG1/2 promoter region -117G.
- the -117G>A mutation is shown in red
- the core sequence of the transcription factor BCL11A binding site is represented by a box
- the PAM sequence is blue.
- the G>A transformation destroys the transcription repressor BCL11A binding site and activates HUDEP- 2 Expression of HBG1/2 in ( ⁇ G ⁇ ).
- Figure 14 is a comparison of the C to T base editing efficiency (ie, the ordinate, the unit is %) achieved by targeting the HBG-117G target in HEK293T cells by hyeA3A-BE4max, eA3A-BE4max, A3A-BE4max, and hyA3A-BE4max.
- Figure 15 is a schematic diagram of the construction of lentiviral vectors Lenti-117G-hyA3A-BE4max-P2A-GFP and Lenti-117G-hyeA3A-BE4max-P2A-GFP.
- the target sequence of sgRNA is HBG-117G.
- FIG 16 is a hyeA3A-BE4max targeting and hyA3A-BE4max C HBG-117G targets implemented in HUDEP-2 ( ⁇ G ⁇ ) cells to T bases editing efficiency (i.e. the ordinate, the unit is%) contrast.
- FIG 17 is a lentivirus infection Lenti-117G-hyeA3A-BE4max- P2A-GFP , and ( ⁇ G ⁇ ) Globin mRNA after cell differentiation Comparative Lenti-117G-hyA3A-BE4max- P2A-GFP in HUDEP-2. Among them, **** means the difference significance level P ⁇ 0.0001.
- Figure 18 is a schematic diagram of the animal model construction of hyA3A-BE4max targeting Duchenne muscular dystrophy (DMD) gene.
- DMD Duchenne muscular dystrophy
- Figure 19 is a comparison of F0 high-throughput sequencing results after microinjection of A3A-BE4max and hyA3A-BE4max.
- Figure 20 shows the average ratio of Reads containing TAA stop codons in F0 produced by A3A-BE4max and hyA3A-BE4max injections.
- FIG. 21 shows the F0 mice produced by immunofluorescence staining to detect the expression of dystrophin (Dystrophin).
- FIG 22 shows the germline inheritance of DMD mutant mice (F0 ⁇ F1).
- Figure 23 is an off-target analysis of the combination of predicted off-target sites of hyA3A-BE4max and DMD-sg3 on the F0 generation.
- the DNA of the targets EMX1 site1 and Tim3-sg1 shown in Table 2 were artificially synthesized and respectively connected to the BbsI site of the sgRNA expression plasmid U6-sgRNA-EF1 ⁇ -GFP (used to express the sgRNA of the corresponding target) to obtain the recombinant plasmid pE And pT.
- Target name Sequence SEQ ID No. EMX1 site1 GAGTCCGAGCAGAAGAAGAAGGG 17 Tim3-sg1 TTCTACACCCCAGCCGCCCCAGG 18 VEGFA site2 GACCCCCTCCACCCCGCCTCCGG 19 Lag3-sg2 CGCTACACGGTGCTGAGCGTGGG 20 HEK3 GGCCCAGACTGAGCACGTGATGG twenty one HEK4 GGCACTGCGGCTGGAGGTGGGGG twenty two EMX1-sg2p GACATCGATGTCCTCCCCATTGG twenty three Nme1-sg1 AGGGATCGTCTTTCAAGGCGAGG twenty four
- each plasmid combination is transfected Set 3 wells to repeat, 2 ⁇ 10 5 cells per well. At the same time, a blank control that does not transfect any plasmid is set.
- pssDBD-BE4max represents: plasmid pRPA70-A-BE4max, pRPA70-B-BE4max, pRPA70-AB-BE4max, pRPA70-C-BE4max, pRPA32-D-BE4max, pBRCA2-OB2-BE4max, pBRCA2-OB3-BE4max, pKH-
- the plasmid pCMV-BE4max is used as a negative control.
- BE4max (Rad51DBD-N-BE4max or Rad51DBD-BE4max) fused with the functional domain of Rad51 single-stranded DNA binding protein has the most significant increase in the editing efficiency of C to T on the target, followed by fusion BE4max of the functional domain of RPA70-C single-stranded DNA binding protein.
- Rad51 DBD was fused to the other two different positions of BE4max, and Rad51DBD was fused.
- BE4max (the third to fifth figures from top to bottom in Figure 1), a total of three recombinant plasmids and recombinant plasmid pE or pT were transfected into cells according to the method 1.2 in step 1, and according to 1.3 and The 1.4 method obtains the editing efficiency results (Table 4 and Table 5).
- Rad51DBD-N-BE4max In BE4max, Rad51 DBD is fused between NLS and rA1, that is, Rad51 DBD is located at the N end of rA1 and spCas9n;
- Rad51DBD-C-BE4max Fusion Rad51 DBD between spCas9n and UGI in BE4max, that is, Rad51 DBD is located at the C end of rA1 and spCas9n;
- hyBE4max fusion of Rad51 DBD between rA1 and spCas9n in BE4max.
- VEGFA site2 Lag3-sg2, HEK3, HEK4, EMX1-sg2p, Nme1-sg1 (sequences shown in Table 2)
- VEGFA site2 Lag3-sg2, HEK3, HEK4, EMX1-sg2p, Nme1-sg1 (sequences shown in Table 2)
- EMX1-sg2p Nme1-sg1 (s shown in Table 2)
- plasmid U6-sgRNA- At the BbsI site of EF1 ⁇ -GFP, recombinant plasmids pV, pL, pH3, pH4, pEP and pN were obtained.
- the plasmid was sequenced by sanger to ensure that it was completely correct.
- Rad51-DBD was synthesized according to the coding sequence in Table 1, and then seamlessly cloned and assembled into the plasmid pCMV-A3A-BE4max expressing the protein A3A-BE4max ( Figure 5) between hA3A and spCas9n to construct the expression fusion protein
- the recombinant plasmid pA of hyA3A-BE4max Figure 5).
- the target sequences are shown in Table 2, FANCF site1, EGFR-sg5, EGFR
- the target sequence of -sg21 is shown in Table 6; respectively connected to the BbsI site of the sgRNA expression plasmid pU6-sgRNA-EF1 ⁇ -GFP to obtain recombinant plasmids pB1, pB2, ... pB8 expressing the sgRNA of the corresponding target.
- Target name Sequence SEQ ID No. FANCF site1 GGAATCCCTTCTGCAGCACCTGG 25 EGFR-sg5 GTGCTGGGCTCCGGTGCGTTCGG 26 EGFR-sg21 CAAAGCAGAAACTCACATCGAGG 27
- step 1.3 where the identification primers of FANCF site1, EGFR-sg5, and EGFR-sg21 are shown in Table 7, and the remaining expressed identification primers are shown in Table 3.
- the fusion protein hyA3A-BE4max had significantly improved editing efficiency of a single base C to T at different positions (C3-C15) of each target ( Figure 6).
- the high activity window of hyA3A-BE4max has expanded from C3-C11 to C3-C15; among them, in C3-C11 far from the PAM region, the editing efficiency of hyA3A-BE4max for a single base C to T is 1.1-2.3 times that of A3A-BE4max.
- the editing efficiency of hyA3A-BE4max for a single base C to T is 3.1-4.1 times that of A3A-BE4max, that is, at C12-C15 near the PAM region , HyA3A-BE4max improves the editing efficiency of a single base C to T more significantly (Figure 7). And hyA3A-BE4max also maintained low indels (Figure 8).
- Rad51-DBD was synthesized according to the coding sequence in Table 1, and then seamlessly cloned and assembled into the plasmid pCMV-eA3A-BE4max expressing the protein eA3A-BE4max ( Figure 9) between eA3A and spCas9n to construct the expression fusion protein hyeA3A-BE4max ( Figure 9)
- the recombinant plasmid pAe The recombinant plasmid pAe.
- EMX1-sg2p Simultaneously designed and synthesized 11 human endogenous targets: EMX1-sg2p, EMX1 site1, Nme1-sg1.
- the target sequences are shown in Table 2, and the target sequences of EGFR-sg21 are shown in Table 6, and the rest of the targets The sequence is shown in Table 8, respectively connected to the BbsI site of the sgRNA expression plasmid U6-sgRNA-EF1 ⁇ -GFP to express the sgRNA of the corresponding target to obtain the recombinant plasmid pC1, pC2, ... pC11.
- Target name Sequence (5 ⁇ -3 ⁇ ) SEQ ID No. CTLA-sg1 CTCCCTCAAGCAGGCCCCGCTGG 28 EGFR-sg5 GTGCTGGGCTCCGGTGCGTTCGG 29 CDK10-sg1 TTCTCGGAGGCTCAGGTGCGTGG 30 EMX1-sg1 GCTCCCATCACATCAACCGGTGG 31 HPRT1-sg6 GCCCTCTGTGTGCTCAAGGGGGG 32 EGFR-sg26 CATGCCCTTCGGCTGCCTCCTGG 33 CCR5-sg1 TAATAATTGATGTCATAGATTGG 34
- step 1.3 where the identification primers of EMX1-sg2p, EMX1 site1, Nme1-sg1 are shown in Table 3, the identification primers of EGFR-sg21 are shown in Table 7, and the remaining target sequences are shown in Table 9.
- the fusion protein hyeA3A-BE4max has a significant increase in the editing efficiency of a single base C to T at different positions (C3-C15) of each target site, and the high activity window is expanded from the original C3-C11 To C3-C15, it can specifically target a single base C in the TC motif to achieve C to T conversion (Figure 10); among them, at C3-C11 far away from the PAM region, hyeA3A-BE4max pairs a single base C to T.
- the editing efficiency of eA3A-BE4max is 1.6-2.8 times that of eA3A-BE4max.
- the editing efficiency of hyeA3A-BE4max for a single base C to T is 4.5-31.9 times that of eA3A-BE4max, that is, the editing efficiency of hyeA3A-BE4max is 4.5-31.9 times that of eA3A-BE4max.
- the editing efficiency of single base C to T is improved more obviously ( Figure 11).
- hyeA3A-BE4max kept low indels ( Figure 12).
- Method 6 performs deep sequencing and statistical analysis.
- the construction method of the above recombinant plasmid pC12 is as follows: the sgRNA target sequence of HBG-117G (GGCTATTGGTCAAGGCAAGGCTGG, SEQ ID No. 35) is connected to the BbsI site of the sgRNA expression plasmid U6-sgRNA-EF1 ⁇ -GFP to express the corresponding target Spot the sgRNA to obtain the recombinant plasmid pC12.
- the identification primers used for the above-mentioned deep sequencing target HBG-117G are as follows:
- HBG-117G F AGTGAGTACGGTGTGCTGGAATGACTGAATCGGAACAAGGC;
- HBG-117G R GTTGGATGCTGGATGGCTGGCCTCACTGGATACTCTAAGACT.
- A3A-BE4max not only targets the -117G>A(C11) mutation in the HBG1/2 promoter region, but also produces -109G>A(C3), -122G>A(C16) "Bystander" mutation; for eA3A-BE4max and hyeA3A-BE4max, eA3A-BE4max accurately edits the G to A transition on -117 (that is, the complementary chain C to T transition) without causing the "bystander" mutation, But the efficiency is very low.
- hyeA3A-BE4max increases the G to A conversion efficiency by 6.6 times, and there is no detectable "bystander” mutation at -109 and -122.
- the results show that hyeA3A-BE4max exhibits precise and efficient targeting of -117G in the HBG1/2 promoter region (a summary of the mechanism of action is shown in Figure 13).
- the hyA3A-BE4max coding sequence was seamlessly cloned to replace BE3 on the backbone vector to obtain the lentiviral vector Lenti-hyA3A-BE4max-P2A-GFP.
- the hyeA3A-BE4max coding sequence was seamlessly cloned to replace BE3 on the backbone vector to obtain the lentiviral vector Lenti-hyeA3A-BE4max-P2A-GFP.
- the virus supernatant was collected from HEK293T cell supernatant at 48h after transfection (starting at 0 hours after transfection) and 72h. Centrifuge at 4000g for 10 min at 4°C to remove cell debris, then filter the supernatant with a 0.45 ⁇ m filter in a 40mL ultrafiltration centrifuge tube, add the crude lentivirus extract to the filter cup, and centrifuge at 25000g at 4°C for 2.5 hours. After centrifugation, take out the centrifugal device and separate the filter cup and the filtrate collection cup below.
- the liquid in the sample collection cup is the virus concentrate (containing the lentivirus Lenti-117G-hyA3A-BE4max-P2A-GFP or the lentivirus Lenti-117G-hyeA3A-BE4max-P2A-GFP). Remove the virus concentrate and store it in virus tubes after aliquoting at -80°C for long-term storage.
- the HUDEP-2 ( ⁇ G ⁇ ) cells infected with the lentivirus Lenti-117G-hyA3A-BE4max-P2A-GFP or Lenti-117G-hyeA3A-BE4max-P2A-GFP were flow-sorted for GFP-positive cells and cultured until the number of cells was greater than 5 After ⁇ 10 4 , the cells are collected, genomic DNA is extracted, and deep sequencing and analysis are performed according to the method in step 5.1.
- HUDEP-2 ( ⁇ G ⁇ ) cells infected with the lentivirus Lenti-117G-hyA3A-BE4max-P2A-GFP or Lenti-117G-hyeA3A-BE4max-P2A-GFP were flow-sorted for GFP-positive cells and cultured until the number of cells was greater than 5 ⁇ 10 4 , after about 5-7 days, HUDEP-2 ( ⁇ G ⁇ ) cells were collected for differentiation and expression.
- the differentiation process is as follows:
- HUDEP-2 ( ⁇ G ⁇ ) cells were differentiated in erythrocyte-like differentiation medium (IMDM), supplemented with 2% human blood type AB plasma (serum) (Gemini, 100-512), 1% L-glutamine, 2IU/mL heparin, erythropoietin (EPO, 3IU/ml, PeproTech), 330 ⁇ g/mL Holo-human transferrin (Sigma-Aldrich), human stem cell factor (SCF, 50ng/ ml, PeproTech), 2% Pen/Strep (Gibco), 10 ⁇ g/ml recombinant human insulin, differentiation for 8 days.
- IMDM erythrocyte-like differentiation medium
- ⁇ -globin expression detection 8 days after differentiation, the cells were harvested and the total mRNA was extracted by phenol-chloroform extraction method.
- the primers (5’-3’) are as follows:
- HBG-QPCR-F GGTTATCAATAAGCTCCTAGTCC;
- HBG-QPCR-R ACAACCAGGAGCCTTCCCA
- HBB-QPCR-F TGAGGAGAAGTCTGCCGTTAC
- HBB-QPCR-F ACCACCAGCAGCCTGCCCA.
- hyA3A-BE4max and hyeA3A-BE4max can significantly increase the level of ⁇ -globin mRNA in HUDEP-2 ( ⁇ G ⁇ ) cells; hyeA3A-BE4max has a positive effect on HUDEP
- the level of ⁇ -globin mRNA increased in -2 ( ⁇ G ⁇ ) cells was three times that of hyA3A-BE4max ( Figure 17).
- mice used below are C57/BL6 mice.
- T7 templates of A3A-BE4max and hyA3A-BE4max were transcribed in vitro to obtain working system mRNA and purified.
- mRNA containing A3A-BE4max or mRNA containing hyA3A-BE4max a working system mRNA (mRNA containing A3A-BE4max or mRNA containing hyA3A-BE4max) with a total volume of 20 ⁇ L and a final concentration of 100ng/ ⁇ L and sgRNA with a final concentration of 200ng/ ⁇ L (DMD-sg3) mixed solution.
- the superovulated donor mother mice were killed by carbon dioxide asphyxiation method, and the oviducts were taken out, placed in a petri dish, and preheated M2 medium was added to the petri dish.
- the target-site DMA-sg3 primer pair F/R (Table 11) was used to obtain the target-containing DNA fragments.
- high-throughput results among the 10 F0 in the hyA3A-BE4max treatment group, there were 6 homozygous terminating nonsense mutations from CAA to TAA (numbered #BD03, #BD05, # in the bottom figure in Figure 19).
- BD07, #BD12, #BD15, #BD16 mice) and no termination nonsense mutation from CAA to TAA was found in the A3A-BE4max treatment group F0 ( Figure 19 upper panel).
- mice in the WT(+/+) and A3A-BE4max treatment groups (such as #AD26)
- only the mice in the hyA3A-BE4max treatment group caused homozygous C to T at position 10 of the DMA-sg3 target sequence DMD in the 6 F0 generation mice with nonsense mutations (#BD03 in Figure 21) did not get protein expression (Figure 21), which also proved that the DMD animal disease model was successfully constructed.
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Abstract
提供一种提高基因编辑效率的融合蛋白及其应用。所述融合蛋白包括单链DNA结合蛋白功能域、核苷脱氨酶和核酸酶。根据CBEs行使C-G到T-A碱基转换过程中,核苷脱氨酶如胞嘧啶脱氨酶以单链DNA为底物进行脱氨,通过将核苷脱氨酶和核酸酶的融合蛋白上再融合单链DNA结合蛋白功能域,大大增加了单链DNA暴露于核苷脱氨酶的机会,从而明显提高了碱基编辑效率。对单碱基基因编辑技术进行了突破性的改进,可以极大地促进其在基因编辑、基因治疗、细胞治疗、动物模型制作、作物遗传育种等方面的应用。
Description
本发明涉及生物技术领域,具体说是一种提高基因编辑效率的融合蛋白及其应用。
自2013年以来,以CRISPR/Cas9为代表的新一代基因编辑技术进入生物学领域的各个实验,改变了传统的基因操作手段。2016年4月,David Liu实验室首次报导单碱基基因编辑技术,之后,基于胞嘧啶脱氨酶原理的其它类型的单碱基基因编辑技术(如来源于七鳃鳗和人的胞嘧啶脱氨酶以不同方式与dCas9或Cas9n融合)也相继被报道。它以CRISPR/Cas9中来源于酿脓链球菌(Streptococcus pyogenes)spCas9以NGG作为PAM(间隔序列前体临近基序)并识别和特异结合DNA在NGG上游实现C到T或G到A的单碱基突变。
单碱基基因编辑技术,已被报导可用于高效地进行基因组的基因突变或修复、疾病动物模型制作、基因治疗。目前,已发现的单碱基基因编辑工具中,以BE3(碱基编辑器3)应用最为广泛。BE3以最高达37%的碱基替换效率,远远高于利用同源重组实现的效率,同时保持较低的脱靶效应,展现出其在用于基因组的单碱基突变修饰或单碱基突变治疗的巨大潜力。随着研究的深入,发现引入额外两个或者更多拷贝的UGI(尿嘧啶糖苷酶抑制剂)到BE3可进一步增强其编辑效率及产物纯度。引入双分型NLS(核定位信号)和密码子化即BE4max,其编辑效率被一步提高。这些方法均一定程度提高其效率,但都比较有限。
β-血红蛋白病如β地中海贫血和镰状细胞病(SCD),是由编码β血红蛋白基因HBB突变引起的。在极少数情况下,遗传性胎儿血红蛋白病(HPFH)是一种良性遗传性疾病,成年病人体内高表达γ-珠蛋白可以缓解因β血红蛋白突变而产生的疾病表型。已报导用CRISPR/Cas9删除HBG1/2的启动子区13bp可以激活γ-球蛋白表达,进而缓解或治疗地中海贫血疾病,是一种有效地治疗策略。据报道,在其中一位HPFH患者中,HBG1/2启动子区-117位点的杂合点突变(G>A)产生10-20%的胎儿血红蛋白(HbF)表达,其机制是-117G>A的突变破坏了转录抑制因子BCL11A的结合位点。
现有基因编辑技术利用CRISPR/Cas9介导的同源重组制备因碱基替代而产生动物模型的效率还比较低。新型的单碱基基因编辑技术以100%的效率产生疾病动物模型而备受关注,然而现有单碱基基因编辑技术通常是C3-C8,靶向靠近PAM区C不是很有效。
发明内容
针对现有技术中存在的缺陷,本发明的目的在于提供一种提高基因编辑效率的融合蛋白及其应用。
一方面,本发明提供了一种提高基因编辑效率的融合蛋白,包括单链DNA结合蛋白功能域、核苷脱氨酶和核酸酶。
具体的,所述融合蛋白的连接顺序为:所述核苷脱氨酶位于所述核酸酶的N端或C端,所述单链DNA结合蛋白功能域位于所述核苷脱氨酶和所述核酸酶的N端、C端和/或所述核苷脱氨酶和所述核酸酶之间;
优选的,所述核苷脱氨酶位于所述核酸酶的N端;
更优选的,所述单链DNA结合蛋白功能域位于所述核苷脱氨酶和所述核酸酶之间。
在上述融合蛋白中,所述单链DNA结合蛋白包括序列特异性单链DNA结合蛋白、和/或非序列特异性单链DNA结合蛋白,优选,非序列特异性单链DNA结合蛋白,
优选的,所述非序列特异性单链DNA结合蛋白选自RPA70(人复制蛋白A的70亚基)、 RPA32(人复制蛋白A的32亚基)、BRCA2(乳腺癌2号基因)、hnRNPK(异质核核糖核蛋白K)、PUF60(聚-U结合剪接因子60KDa)和Rad51(一种同源重组修复蛋白)中的任一种或任几种;
优选的,所述序列特异性单链DNA结合蛋白选自TEBP(端粒结合蛋白)、Teb1(一种端粒酶的组成蛋白)和POT1(人端粒保护蛋白1)中的任一种或任几种;
优选的,所述单链DNA结合蛋白功能域包括如下四种结构域中的至少一种(任一种、任两种、任三种或全部四种)或如下四种结构域中具有与单链DNA结合功能的部分多肽片段及其任意组合:所述单链DNA结合蛋白的OB折叠(寡核苷酸/寡糖/寡肽结合折叠)、KH结构域(K同源结构域)、RRMS(RNA识别基序)、涡状结构域(whirly domains);
更优选的,所述单链DNA结合蛋白功能域包括Rad51的DNA结合结构域(DBD),更优选的,所述Rad51的DNA结合结构域的氨基酸序列包括SEQ ID No.1所示序列,更优选的,所述Rad51的DNA结合结构域的编码序列包括SEQ ID No.2所示序列;
更优选的,所述RPA70的DNA结合结构域的氨基酸序列包括SEQ ID No.11所示序列,更优选的,所述RPA70的DNA结合结构域的编码序列包括SEQ ID No.12所示序列。
在上述融合蛋白中,所述脱氨酶包括胞嘧啶脱氨酶(APOBEC)和/或腺苷脱氨酶,优选,胞嘧啶脱氨酶,所述胞嘧啶脱氨酶可以来源于不同的生物体,
在一些实施方式中,所述胞嘧啶脱氨酶包括来源于大鼠的胞嘧啶脱氨酶,优选的,所述来源于大鼠的胞嘧啶脱氨酶氨基酸序列包括SEQ ID No.3所示序列,更优选的,所述来源于大鼠的胞嘧啶脱氨酶的编码序列包括SEQ ID No.4所示序列;
在一些实施方式中,所述胞嘧啶脱氨酶包括来源于人的胞嘧啶脱氨酶APOBEC3A,优选的,所述来源于人的胞嘧啶脱氨酶APOBEC3A的氨基酸序列包括SEQ ID No.13所示序列,更优选的,所述胞嘧啶脱氨酶APOBEC3A的编码序列包括SEQ ID No.14所示序列;
在一些实施方式中,所述胞嘧啶脱氨酶包括胞嘧啶脱氨酶APOBEC3A突变体,所述胞嘧啶脱氨酶APOBEC3A突变体是将所述胞嘧啶脱氨酶APOBEC3A第57位(自起始密码子)的天冬酰胺(N或Asn)突变为甘氨酸(G或Gly),优选的,该胞嘧啶脱氨酶APOBEC3A来源于人,更优选的,该胞嘧啶脱氨酶APOBEC3A的氨基酸序列包括SEQ ID No.13所示序列,更优选的,该胞嘧啶脱氨酶APOBEC3A的编码序列包括SEQ ID No.14所示序列;所述胞嘧啶脱氨酶APOBEC3A突变体的氨基酸序列包括SEQ ID No.15所示序列,更优选的,所述胞嘧啶脱氨酶APOBEC3A的编码序列包括SEQ ID No.16所示序列;
在上述融合蛋白中,所述核酸酶选自Cas9、Cas3、Cas8a、Cas8b、Cas10d、Cse1、Csy1、Csn2、Cas4、Cas10、Csm2、Cmr5、Fok1、Cpf1中的一种或任意几种;优选的,所述核酸酶为Cas9;更优选的,所述Cas9选自来源于肺炎链球菌、金黄色葡萄球菌、酿脓链球菌或嗜热链球菌的Cas9,更优选的,所述Cas9选自Cas9突变体VQR-spCas9、VRER-spCas9、spCas9n,更优选,spCas9n,更优选的,所述spCas9n的氨基酸序列包括SEQ ID No.5所示序列,更优选的,所述spCas9n的编码序列包括SEQ ID No.6所示序列。
在上述融合蛋白中,还包括NLS(核定位信号),优选的,所述NLS位于所述融合蛋白的至少一端(C端和/或N端);更优选的,所述NLS的氨基酸序列包括SEQ ID No.7所示序列,更优选的,所述NLS的编码序列包括SEQ ID No.8所示序列;
所述融合蛋白还包括UGI(尿嘧啶糖苷酶抑制剂),优选的,所述UGI位于所述融合蛋白的至少一端(C端和/或N端);更优选的,所述UGI的氨基酸序列包括SEQ ID No.9所示序 列,更优选的,所述UGI的编码序列包括SEQ ID No.10所示序列,更优选的,所述UGI为两个以上拷贝。
另一方面,本发明还提供了如下A)-C)生物材料中的任一种:
A)一种基因,编码以上任一所述的融合蛋白;所述基因为DNA或RNA(如mRNA);
B)一种重组载体,含有A)所述基因;所述重组载体包括病毒载体、和/或非病毒载体;所述病毒载体包括腺相关病毒载体、腺病毒载体、慢病毒载体、逆转录病毒载体、和/或溶瘤病毒载体,所述非病毒载体包括阳离子高分子聚合物、质粒载体和/或脂质体;
C)一种重组细胞或重组菌,含有以上任一所述的融合蛋白,或含有A)所述基因,所述重组菌可为工程菌,所述重组细胞可为待编辑的目的细胞,如免疫细胞(如T细胞)、造血干细胞、骨髓细胞、血红细胞,优选,红细胞前体细胞或造血干细胞。
另一方面,本发明还提供了一种对细胞内目的基因进行基因编辑的sgRNA,所述sgRNA的靶序列包括SEQ ID No.17-36中的至少一种,
优选的,所述细胞为T细胞、造血干细胞、骨髓细胞或血红细胞,更优选,红细胞前体细胞或造血干细胞,
优选的,所述目的基因为HBG1和HBG2启动子区(具体为-117位点的G编辑为A,-117位点的G即所述启动子区CCAGCCTTGCCTTGACCAATAGCC中左起第14位G)。
另一方面,本发明还提供了一种单碱基基因编辑系统,包括以上任一所述的融合蛋白或所述的生物材料和sgRNA,所述sgRNA引导所述融合蛋白对目的细胞中的目的基因进行单碱基基因编辑;
优选的,所述sgRNA的靶序列包括SEQ ID No.17-36中至少一种;
和/或,所述细胞为T细胞、造血干细胞、骨髓细胞、血红细胞、或红细胞前体细胞,
和/或,所述目的基因为HBG1和HBG2启动子区。
另一方面,本发明保护以上任一所述融合蛋白、所述的生物材料、所述的单碱基基因编辑系统在制备基因编辑产品、疾病治疗和/预防产品、动物模型、或植物新品种中的应用;
在一些实施例中,所述疾病为β血红蛋白病,所述β血红蛋白病包括β地中海贫血和/或镰刀型细胞贫血症。
另一方面,本发明还提供了一种提高单碱基基因编辑效率的方法,包括利用以上任一所述的融合蛋白和sgRNA引入细胞、对目的基因进行基因编辑的步骤,所述sgRNA引导所述融合蛋白对所述目的基因进行单碱基基因编辑。
在上述方法中,优选的,所述sgRNA的靶序列包括SEQ ID No.17-36中的至少一种;
和/或,所述细胞为T细胞、造血干细胞、骨髓细胞、血红细胞、或红细胞前体细胞;
和/或,所述目的基因为HBG1和HBG2启动子区。
另一方面,本发明还提供了一种疾病动物模型的构建方法,包括利用以上任一所述的融合蛋白和sgRNA引入动物细胞、对目的基因进行基因编辑的步骤;
优选的,所述sgRNA的靶序列包括SEQ ID No.17-36中的至少一种;更优选的,所述sgRNA的靶序列包括SEQ ID No.36所示序列,所述目的基因包括DMD基因;
优选的,所述动物为哺乳动物,更优选的,所述哺乳动物为大鼠或小鼠,更优选小鼠;
优选的,所述细胞为胚胎细胞;
优选的,所述引入的方式为载体转化、显微注射、转染、脂质转染、热休克、电穿孔、转导、基因枪、DEAE-葡聚糖介导的转移中的一种或任几种组合,更优选,显微注射;
优选的,所述引入使用以上任一所述的融合蛋白的mRNA和所述sgRNA进行,
更优选的,所述引入使用的以上任一所述的融合蛋白的mRNA的浓度为1-1000ng/μL,更优选,10-600ng/μL,更优选,50-150ng/μL,更优选,100ng/μL,所述引入使用的所述sgRNA的浓度为1-1000ng/μL,更优选,10-600ng/μL,更优选,150-250ng/μL,更优选,200ng/μL,
更优选的,所述引入使用的以上任一所述融合蛋白的mRNA与所述引入使用的所述sgRNA的浓度比为1:(5-1),更优选,1:(4-1.5),更优选,1:(3-1.8),更优选,1:2。
本发明保护所述方法得到的动物模型在药物筛选、疾病治疗效果评价或疾病治病机理研究中的应用。
另一方面,本发明提供了一种用于治疗和/或预防β血红蛋白病的产品,包含:以上A)中所述基因和sgRNA的递送载体,
所述sgRNA引导所述融合蛋白对目的细胞中HBG1和HBG2启动子区(具体为-117位点的G编辑为A,-117位点的G即所述启动子区CCAGCCTTGCCTTGACCAATAGCC中左起第14位G)进行单碱基基因编辑;
优选的,所述sgRNA的靶序列包括SEQ ID No.35所示序列;
优选的,所述β血红蛋白病包括β地中海贫血和/或镰刀型细胞贫血症;
优选的,所述细胞为T细胞、造血干细胞、骨髓细胞或血红细胞,更优选,红细胞前体细胞或造血干细胞,
优选的,所述目的基因为HBG1和HBG2启动子区(具体为-117位点的G编辑为A,-117位点的G即所述启动子区CCAGCCTTGCCTTGACCAATAGCC中左起第14位G)。
在上述产品中,所述递送载体包括病毒载体、和/或非病毒载体;所述病毒载体包括腺相关病毒载体、腺病毒载体、慢病毒载体、逆转录病毒载体、和/或溶瘤病毒载体,所述非病毒载体包括阳离子高分子聚合物、质粒载体和/或脂质体;
优选的,所述递送载体包括慢病毒载体。
以上所述“至少一种”为其限定的所有种类中的任一种、任两种组合、任三种组合、……、或所有种类组合,均在本申请的保护范围之内。
以上所述氨基酸序列或编码序列中,与本申请所述序列的同源性在80%以上、85%以上、90%以上、95%以上、96%以上、97%以上、98%以上、或99%以上的序列,和/或在本申请所述序列的基础上进行氨基酸残基或核苷酸的替换、删除或插入后的序列,且具有本申请所使用序列具有相同或相近功能的序列,均在本申请的保护范围之内。
本发明的有益效果如下:
本发明根据CBEs(嘧啶碱基转换技术)行使C-G到T-A碱基转换过程中,核苷脱氨酶如胞嘧啶脱氨酶以单链DNA为底物进行脱氨,通过将核苷脱氨酶和核酸酶的融合蛋白上再融合单链DNA结合蛋白功能域,大大增加了单链DNA暴露于核苷脱氨酶的机会,从而明显提高了碱基编辑效率。
本发明通过筛选10种非序列偏好的单链DNA结合蛋白的功能域与BE4max进行融合,发现来源于人的Rad51的一个单链DNA结合功能域(1-114AA)融合在Apobec1与Cas9n的中间展示出最高效率提高,命名为hyBE4max。相对于BE4max,hyBE4max的C-G到T-A编辑效率最大提高了16倍,尤其是靠近PAM区的位点效率提高更为明显,同时保持较低的indels(插入或缺失)。
本发明对单碱基基因编辑技术进行了突破性的改进,可以极大地促进其在基因编辑、基因 治疗、细胞治疗、动物模型制作、作物遗传育种等方面的应用。
本发明在基因治疗中,以β-血红蛋白病为例,相对于eA3A-BE4max,A3A-BE4max,hyeA3A-BE4max靶向HBG1和HBG2(以下简称HBG1/2)启动子区较靠近PAM区-117位点,能更加精确高效地靶向-117产生G到A的突变,从而激活γ-珠蛋白的表达,为临床治疗β-血红蛋白病提供了一种更加精准高效的治疗策略。
本发明将hyA3A-BE4max运用到制作小鼠疾病动物模型中,相对于A3A-BE4max,hyA3A-BE4max靶向靠近PAM区的C到T的突变产生疾病动物模型更加有效,因此本发明提供了的一个新型的高效产生疾病动物模型制作的平台,将极大地促进不同动物模型的生产进程。
图1为不同单链DNA结合蛋白功能域与BE4max融合的结构示意图。其中,NLS为核定位信号(其氨基酸序列如SEQ ID No.7所示,编码序列如SEQ ID No.8所示),rA1为胞苷脱氨酶APOBEC1(其氨基酸序列如SEQ ID No.3所示,编码序列如SEQ ID No.4所示),spCas9n为源于酿脓链球菌(Streptococcus pyogenes)的Cas9n(其氨基酸序列如SEQ ID No.5所示,编码序列如SEQ ID No.6所示),UGI为尿嘧啶糖苷酶抑制剂(其氨基酸序列如SEQ ID No.9所示,编码序列如SEQ ID No.10所示),SSDBD为一种单链DNA结合蛋白功能域。
图2为hyBE4max与BE4max在293T上8个靶点实现的C到T碱基编辑效率(即纵坐标,单位为%)对比。
图3为hyBE4max与BE4max在293T上8个靶点产生的平均C到T碱基编辑效率(即纵坐标,单位为%)对比。
图4为hyBE4max与BE4max在293T上8个靶点产生的indels的碱基编辑效率(即纵坐标,单位为%)对比。
图5为融合蛋白A3A-BE4max和hyA3A-BE4max的结构示意图。其中,hA3A为来源于人的胞苷脱氨酶APOBEC3A(其氨基酸序列如SEQ ID No.13所示,编码序列如SEQ ID No.14所示),NLS、spCas9n和UGI同图1。
图6为hyA3A-BE4max与A3A-BE4max在293T上8个内源性靶点实现的C到T碱基编辑效率(即纵坐标,单位为%)的对比。
图7为hyA3A-BE4max与A3A-BE4max在293T上8个内源性靶点实现的C到T碱基编辑效率(即纵坐标,单位为%)的平均值对比。
图8为hyA3A-BE4max与A3A-BE4max在293T上8个内源性靶点产生的indels的碱基编辑效率(即纵坐标,单位为%)对比。
图9为融合蛋白eA3A-BE4max和hyeA3A-BE4max的结构示意图。其中,A3A N57G为图5中所用hA3A的N57G突变体(其氨基酸序列如SEQ ID No.15所示,编码序列如SEQ ID No.16所示),NLS、spCas9n和UGI同图1。
图10为hyeA3A-BE4max与eA3A-BE4max在293T上11个内源性靶点实现的C到T碱基编辑效率(即纵坐标,单位为%)的对比。
图11为hyeA3A-BE4max与eA3A-BE4max在293T上11个内源性靶点实现的C到T碱基编辑效率(即纵坐标,单位为%)的平均值对比。
图12为hyeA3A-BE4max与eA3A-BE4max在293T上11个内源性靶点产生的indels的碱基编辑效率(即纵坐标,单位为%)对比。
其中,图2、3、5、6、11中横坐标的C及其后面的数字代表被编辑为T的C在相应靶点序列上的位置,如C5代表自相应靶点序列5’端的第5位C被编辑为T的效率。
图13为hyeA3A-BE4max靶向HBG1/2启动子区-117G示意图。其中,-117G>A突变显示为红色,转录因子BCL11A结合位点的核心序列用方框表示,PAM序列为蓝色,G>A转化破坏了转录抑制因子BCL11A的结合位点并激活了HUDEP-2(Δ
Gγ)中HBG1/2的表达。
图14为hyeA3A-BE4max、eA3A-BE4max、A3A-BE4max、hyA3A-BE4max在HEK293T细胞中靶向HBG-117G靶点实现的C到T碱基编辑效率(即纵坐标,单位为%)对比。
图15为慢病毒载体Lenti-117G-hyA3A-BE4max-P2A-GFP和Lenti-117G-hyeA3A-BE4max-P2A-GFP构建示意图。其中,sgRNA的靶点序列为HBG-117G。
图16为hyeA3A-BE4max和hyA3A-BE4max在HUDEP-2(Δ
Gγ)细胞中靶向HBG-117G靶点实现的C到T碱基编辑效率(即纵坐标,单位为%)对比。
图17为感染慢病毒Lenti-117G-hyeA3A-BE4max-P2A-GFP和Lenti-117G-hyA3A-BE4max-P2A-GFP的HUDEP-2(Δ
Gγ)细胞分化后珠蛋白mRNA表达对比。其中,****表示差异显著性水平P<0.0001。
图18为hyA3A-BE4max靶向杜氏肌营养不良(DMD)基因的动物模型构建示意图。
图19为A3A-BE4max和hyA3A-BE4max显微注射后产生F0高通量测序结果的比对。
图20为A3A-BE4max和hyA3A-BE4max注射产生F0中的含有TAA终止密码子的Reads平均比率。
图21为将产生的F0小鼠进行免疫荧光染色检测抗肌萎缩蛋白(Dystrophin)的表达。
图22为DMD突变小鼠的生殖系遗传(F0→F1)。
图23为hyA3A-BE4max与DMD-sg3的预测脱靶位点组合在F0代上的脱靶分析。
一、融合Rad51DBD(1-114aa)单链DNA结合蛋白的功能域的BE4max编辑效率提高最为明显
1.1、质粒设计及构建
1.1.1、根据单碱基编辑技术中CBEs的Apobec1以单链DNA作为底物的特性,我们设计了10个来源于人的非序列偏好性的单链DNA结合蛋白不同的功能域(主要为RPA70(630aa)-A、RPA70-B、RPA70-AB、RPA70-C、RPA32-D、BRCA2-OB2、BRCA2-OB3、HNRNPK KH结构域、PUF60RRM、Rad51 DBD)(表1),由于已报道的融合蛋白放于BE4max(图1中由上至下的第一个图)的C端倾向于没有活性,因此,将上述这些功能域融合在BE4max的N端(图1中由上至下的第二个图),同时设计了两个来自于人的内源性靶点EMX1 site1、Tim3-sg1(序列如表2所示)。
1.1.2、人工合成表1所示10个来源于人的非序列偏好性的单链DNA结合蛋白不同的功能域的DNA,之后进行无缝克隆组装至质粒pCMV-BE4max(addgene,#112093)中BE4max的N端,分别构建了10个重组质粒(图1):pRPA70-A-BE4max、pRPA70-B-BE4max、pRPA70-AB-BE4max、pRPA70-C-BE4max、pRPA32-D-BE4max、pBRCA2-OB2-BE4max、pBRCA2-OB3-BE4max、pKH-BE4max、pRRM-BE4max、pRad51DBD-BE4max。
人工合成表2所示靶点EMX1 site1和Tim3-sg1的DNA,分别连接至sgRNA表达质粒U6-sgRNA-EF1α-GFP的BbsⅠ位点处(用于表达相应靶点的sgRNA),得到重组质粒pE和pT。
1.1.3、将1.1.1与1.1.2中构建的质粒经sanger测序,确保完全正确。
表1、所用的单链DNA结合蛋白不同的功能域序列
表2、所用靶点及序列
靶点名称 | 序列(5’-3’) | SEQ ID No. |
EMX1 site1 | GAGTCCGAGCAGAAGAAGAAGGG | 17 |
Tim3-sg1 | TTCTACACCCCAGCCGCCCCAGG | 18 |
VEGFA site2 | GACCCCCTCCACCCCGCCTCCGG | 19 |
Lag3-sg2 | CGCTACACGGTGCTGAGCGTGGG | 20 |
HEK3 | GGCCCAGACTGAGCACGTGATGG | 21 |
HEK4 | GGCACTGCGGCTGGAGGTGGGGG | 22 |
EMX1-sg2p | GACATCGATGTCCTCCCCATTGG | 23 |
Nme1-sg1 | AGGGATCGTCTTTCAAGGCGAGG | 24 |
1.2、细胞转染
将HEK293T 5×10
5细胞铺24孔板,待细胞长至70%-80%时,按pssDBD-BE4max:pE(或pT)=750ng:250ng进行质粒组合的转染,每种质粒组合转染设3孔重复,每孔2×10
5个细胞。同时设不转染任何质粒的空白对照。
pssDBD-BE4max代表:质粒pRPA70-A-BE4max、pRPA70-B-BE4max、pRPA70-AB-BE4max、pRPA70-C-BE4max、pRPA32-D-BE4max、pBRCA2-OB2-BE4max、 pBRCA2-OB3-BE4max、pKH-BE4max、pRRM-BE4max、pRad51DBD-BE4max中任一种,以质粒pCMV-BE4max为阴性对照。
1.3、基因组提取及扩增子文库的准备
转染后72h,用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hitom试剂盒的操作流程,设计相对应的鉴定引物(表3),即在正向鉴定引物5’端加上搭桥序列5’-ggagtgagtacggtgtgc-3’,反向鉴定引物5’端加上搭桥序列5’-gagttggatgctggatgg-3’,即得到一轮PCR产物,之后利用一轮PCR产物作为模板,进行二轮PCR,后混在一起进行切胶回收纯化后进行送公司进行深度测序。
表3、所用靶点的鉴定引物
1.4、深度测序结果分析与统计
利用BE-analyzer网站分析步骤1.3的深度测序结果,统计C到T、Indels的比率,结果如表4和表5所示。
结果表明:与BE4max相比,融合Rad51单链DNA结合蛋白的功能域的BE4max(Rad51DBD-N-BE4max或Rad51DBD-BE4max),对靶点上的C到T的编辑效率提高最为明显,其次为融合RPA70-C单链DNA结合蛋白的功能域的BE4max。
二、hyBE4max的编辑效率最优
为了进一步测试步骤一中对靶点上的C到T的编辑效率提高最高的Rad51单链DNA结合蛋白功能域的融合位置,将Rad51 DBD融合于BE4max的另两个不同位置,将融合了Rad51DBD的BE4max(图1中的由上至下的第三至五图)共三种重组质粒分别与重组质粒pE或pT按照步骤一中的1.2的方法进行细胞转染,并按照步骤一中的1.3和1.4的方法获得编辑效率结果(表4和表5)。
图1中由上至下的第三至五图所示三种融合了Rad51 DBD的BE4max分别如下:
Rad51DBD-N-BE4max:在BE4max中NLS和rA1之间融合Rad51 DBD,即Rad51 DBD位于rA1和spCas9n的N端;
Rad51DBD-C-BE4max:在BE4max中spCas9n和UGI之间融合Rad51 DBD,即Rad51 DBD 位于rA1和spCas9n的C端;
hyBE4max:在BE4max中rA1和spCas9n之间融合Rad51 DBD。
表4、靶点EMX1 site1的编辑效率结果(单位,%)
表5、靶点Tim3-sg1的编辑效率结果(单位,%)
表4和表5的结果表明:与在BE4max中NLS和rA1之间融合Rad51 DBD(即Rad51DBD-N-BE4max)相比,在BE4max中rA1和spCas9n之间融合Rad51 DBD(即hyBE4max)对靶点上的C到T的编辑效率提高最为明显。
三、hyBE4max的工作特性
为了进一步公平地描述hyBE4max的工作特性,设计另外6个额外的靶点VEGFA site2、Lag3-sg2、HEK3、HEK4、EMX1-sg2p、Nme1-sg1(序列如表2),连接至质粒U6-sgRNA-EF1α-GFP的BbsI位点处,得到重组质粒pV、pL、pH3、pH4、pEP和pN。质粒经sanger测序,确保完全正确。
将步骤二中含有hyBE4max的重组质粒分别与重组质粒pE、pT、pV、pL、pH3、pH4、pEP或pN按照步骤一中的1.2的方法进行细胞转染,并按照步骤一中的1.3和1.4的方法获得编辑效率结果,并用graphpad prism 8.0进行统计作图。
结果如图2和图3所示,在编辑窗口C3-C8,hyBE4max的C到T编辑效率为19-71%,对应的BE4max为13-47%;在编辑窗口C9-C12,hyBE4max的C到T编辑效率为19-55%,对应的BE4max为1.4-17%。相对于BE4max,在编辑窗口C3-C8内,hyBE4max平均C到T编辑效率为BE4max的1.6-2.2倍;在编辑窗口C9-C12内,hyBE4max平均C到T编辑效率为BE4max的3.3-17倍。同时hyBE4max保持较低的indels产生(图4)。
四、含不同胞嘧啶脱氨酶的融合蛋白的效果
(一)、融合蛋白hyA3A-BE4max工作特性
4.1.1、Rad51-DBD按照表1中编码序列进行合成,之后进行无缝克隆组装至表达蛋白A3A-BE4max(图5)的质粒pCMV-A3A-BE4max中hA3A和spCas9n之间,构建表达融合蛋白hyA3A-BE4max(图5)的重组质粒pA。
4.1.2、依次合成8种人的内源性靶点:EMX1 site1、Tim3-sg1、VEGFA site2、EMX1-sg2p、Nme1-sg1的靶序列如表2所示,FANCF site1、EGFR-sg5、EGFR-sg21的靶序列如表6所示;分别连接至sgRNA表达质粒pU6-sgRNA-EF1α-GFP的BbsⅠ位点处,得到表达相应靶点的sgRNA的重组质粒pB1、pB2、……pB8。
4.1.3、将4.1.1与4.1.2中构建的质粒经sanger测序,确保完全正确。
表6、所用靶点及序列
靶点名称 | 序列(5’-3’) | SEQ ID No. |
FANCF site1 | GGAATCCCTTCTGCAGCACCTGG | 25 |
EGFR-sg5 | GTGCTGGGCTCCGGTGCGTTCGG | 26 |
EGFR-sg21 | CAAAGCAGAAACTCACATCGAGG | 27 |
4.1.4、细胞转染
将5×10
5个HEK293T细胞铺24孔板,待细胞长至70%-80%时,按pA(或质粒pCMV-A3A-BE4max):pB1(或pB2、pB3、……pB8)=750ng:250ng进行质粒组合的转染,每种质粒组合转染设3孔重复,每孔2×10
5个细胞。同时设不转染任何质粒的空白对照。
4.1.5、基因组提取及扩增子文库的准备
按照步骤1.3的方法进行,其中,FANCF site1、EGFR-sg5、EGFR-sg21的靶点的鉴定引物如表7所示,其余表达的鉴定引物如表3所示。
表7、所用靶点的鉴定引物
4.1.6、深度测序结果分析与统计
按照步骤1.4的方法进行。
结果表明:相对于蛋白A3A-BE4max,融合蛋白hyA3A-BE4max对各靶点不同位置(C3-C15)单个碱基C到T的编辑效率均明显提高(图6)。相对于A3A-BE4max,hyA3A-BE4max的高活性窗口从原来C3-C11,拓展到C3-C15;其中,在远离PAM区的C3-C11,hyA3A-BE4max对单个碱基C到T的编辑效率是A3A-BE4max的1.1-2.3倍,在靠近PAM区的C12-C15,hyA3A-BE4max对单个碱基C到T的编辑效率是A3A-BE4max的3.1-4.1倍,即在靠近PAM区的C12-C15,hyA3A-BE4max对单个碱基C到T的编辑效率提高更为明显(图7)。且hyA3A-BE4max同时维持较低的indels(图8)。
(二)、融合蛋白hyeA3A-BE4max工作特性
4.2.1、工作系统质粒构建
Rad51-DBD按照表1中编码序列进行合成,之后进行无缝克隆组装至表达蛋白eA3A-BE4max(图9)的质粒pCMV-eA3A-BE4max中eA3A与spCas9n之间,构建表达融合蛋白hyeA3A-BE4max(图9)的重组质粒pAe。
4.2.2、靶点质粒的构建
同时设计并合成11个来自于人的内源性靶点:EMX1-sg2p、EMX1 site1、Nme1-sg1的靶序列如表2所示,EGFR-sg21的靶序列如表6所示,其余靶点序列如表8所示,分别连接至sgRNA表达质粒U6-sgRNA-EF1α-GFP的BbsI位点处,用于表达相应靶点的sgRNA,得到重组质粒pC1、pC2、……pC11。
4.2.3、将4.2.1与4.2.2中构建的质粒经sanger测序,确保完全正确。
表8、所用靶点及序列
靶点名称 | 序列(5`-3`) | SEQ ID No. |
CTLA-sg1 | CTCCCTCAAGCAGGCCCCGCTGG | 28 |
EGFR-sg5 | GTGCTGGGCTCCGGTGCGTTCGG | 29 |
CDK10-sg1 | TTCTCGGAGGCTCAGGTGCGTGG | 30 |
EMX1-sg1 | GCTCCCATCACATCAACCGGTGG | 31 |
HPRT1-sg6 | GCCCTCTGTGTGCTCAAGGGGGG | 32 |
EGFR-sg26 | CATGCCCTTCGGCTGCCTCCTGG | 33 |
CCR5-sg1 | TAATAATTGATGTCATAGATTGG | 34 |
4.2.4、细胞转染-验证hyeA3A-BE4max工作系统
将5×10
5个HEK293T细胞铺24孔板,待细胞长至70%-80%时,按pA(或质粒pCMV-eA3A-BE4max):pC1(或pC2、pC3、……pC11)=750ng:250ng进行质粒组合的转染,每种质粒组合转染设3孔重复,每孔2×10
5个细胞。同时设不转染任何质粒的空白对照。
4.2.5、基因组提取及扩增子文库的准备
按照步骤1.3的方法进行,其中,EMX1-sg2p、EMX1 site1、Nme1-sg1的鉴定引物如表3所示,EGFR-sg21的鉴定引物如表7所示,其余靶点序列如表9所示。
表9、所用靶点的鉴定引物
4.2.6深度测序结果分析与统计
按照步骤1.4的方法进行。
结果表明:相对于蛋白eA3A-BE4max,融合蛋白hyeA3A-BE4max对各靶点不同位置(C3-C15)单个碱基C到T的编辑效率大部分明显提高,且高活性窗口从原来C3-C11拓展到C3-C15位,能特异靶向TC motif中的单个碱基C实现C到T转换(图10);其中,在远离PAM区的C3-C11,hyeA3A-BE4max对单个碱基C到T的编辑效率是eA3A-BE4max的1.6-2.8倍,在靠近PAM区的C12-C15,hyeA3A-BE4max对单个碱基C到T的编辑效率是eA3A-BE4max的4.5-31.9倍,即在靠近PAM区的C12-C15,hyeA3A-BE4max对单个碱基C到T的编辑效率提高更为明显(图11)。同时hyeA3A-BE4max保持较低的indels(图12)。
五、应用融合蛋白hyeA3A-BE4max进行基因治疗
5.1、hyeA3A-BE4max靶向HBG-117G位点在HEK293T细胞上编辑效率的测试
按照4.2.4的细胞转染方法,将按pA(或质粒pCMV-A3A-BE4max、或pAe、或pCMV-eA3A-BE4max):pC12=750ng:250ng进行质粒组合的转染HEK293T细胞;按照4.2.6的方法进行深度测序及统计分析。
上述重组质粒pC12的构建方法如下:将HBG-117G的sgRNA靶点序列(GGCTATTGGTCAAGGCAAGGCTGG,SEQ ID No.35)连接至sgRNA表达质粒U6-sgRNA-EF1α-GFP的BbsI位点处,用于表达相应靶点的sgRNA,得到重组质粒pC12。
上述深度测序靶点HBG-117G所用的鉴定引物如下:
HBG-117G F:AGTGAGTACGGTGTGCTGGAATGACTGAATCGGAACAAGGC;
HBG-117G R:GTTGGATGCTGGATGGCTGGCCTCACTGGATACTCTAAGACT。
结果:如图14所示,A3A-BE4max除能靶向HBG1/2启动子区的-117G>A(C11)突变外,还会产生-109G>A(C3),-122G>A(C16)“旁观者”突变;对于eA3A-BE4max和hyeA3A-BE4max而言,eA3A-BE4max精确地编辑了-117上的G到A转换(即互补链C到T转换)而不引起“旁观者”突变,但效率非常低,与eA3A-BE4max相比,hyeA3A-BE4max提高了6.6倍的G到A转换效率,并且在-109和-122处均未产生可检测到的“旁观者”突变。结果表明,hyeA3A-BE4max展现出精准高效靶向HBG1/2启动子区-117G的特性(作用机理总结如图13所示)。
5.2、慢病毒载体的构建与病毒包装
5.2.1、慢病毒载体的构建
以pLenti-BE3-P2A-Puro(Addgene,#110838)为骨架载体,将hyA3A-BE4max的编码序列通过无缝克隆替换骨架载体上的BE3得到慢病毒载体Lenti-hyA3A-BE4max-P2A-GFP。
以pLenti-BE3-P2A-Puro(Addgene,#110838)为骨架载体,将hyeA3A-BE4max的编码序列通过无缝克隆替换骨架载体上的BE3得到慢病毒载体Lenti-hyeA3A-BE4max-P2A-GFP。
将5.2中HBG-117G靶点序列分别连接至上述两个慢病毒载体的hyA3A-BE4max或hyeA3A-BE4max的上游(图15上图),得到重组质粒Lenti-117G-hyA3A-BE4max-P2A-GFP和重组质粒Lenti-117G hyeA3A-BE4max-P2A-GFP(图15下图)。
5.2.2、慢病毒包装
5.2.2.1、转染
第1天,将生长状态良好的HEK293T细胞消化后铺10cm皿,每个病毒约30皿。第2天,待汇合度80%-90%时,按如下量进行质粒转染:Lenti-117G-hyA3A-BE4max-P2A-GFP(或Lenti-117G-hyeA3A-BE4max-P2A-GFP):PSPAX2:PMD2.G=10ug:10ug:10ug。
5.2.2.2、病毒的收集和纯化
病毒上清收集于转染后48h(转染即可为0小时计起)、72h的HEK293T细胞上清。于4℃,4000g离心10min,除去细胞碎片,后以0.45μm滤器过滤上清液于40mL超滤离心管中,把慢病毒粗提液加入到过滤杯中,4℃、25000g离心2.5小时。离心结束后,取出离心装置,将过滤杯和下面的滤过液收集杯分开。样品收集杯中的液体即为病毒浓缩液(含有慢病毒Lenti-117G-hyA3A-BE4max-P2A-GFP或慢病毒Lenti-117G-hyeA3A-BE4max-P2A-GFP)。将病毒浓缩液移出,分装后保存在病毒管中,-80℃长期保存。
5.3、基因治疗
5.3.1、HUDEP-2(Δ
Gγ)细胞感染病毒
在96孔板铺5×10
4的HUDEP-2(Δ
Gγ)细胞3个孔,总体积为100ul培养基,分别等滴度感染慢病毒Lenti-117G-hyA3A-BE4max-P2A-GFP和Lenti-117G-hyeA3A-BE4max-P2A-GFP,感染体系如下:
5.3.2、编辑效率检测
将感染慢病毒Lenti-117G-hyA3A-BE4max-P2A-GFP或Lenti-117G-hyeA3A-BE4max-P2A-GFP的HUDEP-2(ΔGγ)细胞经流式分选GFP阳性细胞,培养到细胞数量大于5×10
4后,收取细胞,提取基因组DNA,按照步骤5.1的方法进行深度测序和分析。
结果:与hyA3A-BE4max相比,hyeA3A-BE4max有效靶向HUDEP-2(Δ
Gγ)细胞中精确的–117G>A突变,并表现出更高的活性(图16)。
5.3.3分化及γ珠蛋白表达检测
将感染慢病毒Lenti-117G-hyA3A-BE4max-P2A-GFP或Lenti-117G-hyeA3A-BE4max-P2A-GFP的HUDEP-2(ΔGγ)细胞经流式分选GFP阳性细胞,培养到细胞数量大于5×10
4,约5-7天后收集HUDEP-2(Δ
Gγ)细胞进行分化表达。分化过程如下:
计数后,1×10
5个HUDEP-2(Δ
Gγ)细胞分化在类红细胞分化培养基(IMDM)中进行,同时补充2%人血AB型血浆(血清)(Gemini,100-512),1%L-谷氨酰胺,2IU/mL肝素,促红细胞生成素(EPO,3IU/ml,PeproTech),330μg/mL Holo-人类转铁蛋白(Sigma-Aldrich),人干细胞因子(SCF,50ng/ml,PeproTech),2%的Pen/Strep(Gibco),10μg/ml重组人胰岛素,分化8天。
γ珠蛋白表达检测:分化8天后,收细胞用酚氯仿抽提法抽提总的mRNA。使用HiScript II Q RT SuperMix(Vazyme)对分离的mRNA进行逆转录;在QuantiStudio 3实时PCR系统(ABI)上进行qPCR,并通过SYBRGreen qPCR对HBG和HBB mRNA进行定量。引物(5’-3’)如下:
HBG-QPCR-F:GGTTATCAATAAGCTCCTAGTCC;
HBG-QPCR-R:ACAACCAGGAGCCTTCCCA;
HBB-QPCR-F:TGAGGAGAAGTCTGCCGTTAC;
HBB-QPCR-F:ACCACCAGCAGCCTGCCCA。
结果:与HUDEP-2(Δ
Gγ)的WT细胞相比,hyA3A-BE4max和hyeA3A-BE4max可以显著提高HUDEP-2(Δ
Gγ)细胞中γ-珠蛋白mRNA的水平;hyeA3A-BE4max对HUDEP-2(Δ
Gγ)细胞中γ-珠蛋白mRNA水平提高程度是hyA3A-BE4max的3倍(图17)。
六、应用融合蛋白hyA3A-BE4max构建DMD疾病动物模型
以下所用小鼠为C57/BL6小鼠。
6.1、工作系统mRNA与靶点sgRNA转录模板的构建
在NCBI上下载小鼠相关基因序列,如图18所示,在目标位点(抗肌萎缩蛋白基因即DMD基因第12个外显子中矩形框处的位点)设计sgRNA(DMD-sg3靶点序列:ACATCTCATCAAGGACTTGTTGG,SEQ ID No.36),并订购Oligo引物,将通过退火形成的 sgRNA克隆到T7载体骨架中,使用引物对IVT-PCR-F和IVT-PCR-R(表10)体外转录法(IVT)扩增含有T7启动子的DMD-sg3模板,使用引物对IVT-T7-hyA3A-BE4max-F和IVT-T7-hyA3A-BE4max-R(表10)将T7启动子通过PCR引入hyA3A-BE4max或者A3A-BE4max的mRNA模板。
表10、IVT所用PCR引物
6.2、sgRNA(DMD-sg3)体外转录
利用普通DNA产物纯化试剂盒对6.1中PCR产物进行纯化,以纯化后的PCR产物为线性化DNA模板,利用T7体外转录试剂盒(MEGAshortscriptTM Kit)进行体外转录,利用氯化锂沉淀法对转录的sgRNA进行纯化。
6.3、工作系统mRNA(A3A-BE4max和hyA3A-BE4max)的转录
6.4、显微注射混合物的制备
用无核酸酶的水配置注射混合物,获得总体积为20μL,终浓度为100ng/μL的工作系统mRNA(含有A3A-BE4max的mRNA或含有hyA3A-BE4max的mRNA)与终浓度为200ng/μL的sgRNA(DMD-sg3)的混合液。
6.5、一细胞期胚胎的收集
(1)第一天:于下午1-2点间向6-8周龄的供体母鼠腹腔注射100μL(5IU)的PMSG工作液。
(2)第三天:于下午2-4点间向注射过PMSG的母鼠腹腔注射100μL(5IU)的hCG工作液,注射后,将经激素处理后的母鼠与10-14周齡的公鼠一对一合笼。同时,将处于发情期未经激素处理的雌鼠与输卵管结扎的雄鼠在下午4点左右交配用于假孕母鼠的准备。
(3)第四天:早上9点前,检查与结扎公鼠合笼的受体母鼠是否有孕栓,将有孕栓的母鼠集中于新的笼子内用于下午的胚胎移植实验。
(4)通过二氧化碳窒息法将超排卵的供体母鼠处死取出输卵管,放置于平皿中,平皿中加入预热的M2培养基。
(5)将输卵管放置于另一个新的平皿中,平皿内加入预热的M2培养基与透明质酸,M2培养基与透明质酸的体积比为9:1。在体式显微镜下,用摄子拉扯输卵管壶腹部,使胚胎从输卵管释放至平皿中。把胚胎一直孵育在有透明质酸的M2培养基中直到卵丘细胞掉落。在去除卵丘细胞后,将胚胎转移至一个新的平皿中,平皿内加入无透明质酸的M2培养基,反复用M2培养基冲洗胚胎使透明质酸和卵丘细胞均被冲洗干净。
(6)将冲洗干净的胚胎转移至一个新的平皿中,平皿内先分散加入几滴KSOM培养基,然后再缓慢向平皿加入矿物油,使KSOM培养基被矿物油隔开与覆盖。一般来说,在一个35毫米的平皿中,可加6个点的KSOM培养基,每个点为50μL。将50个胚胎作为一组首先放于中间的KSOM培养基点进行漂洗,然后转移至一个新的培养基点。在显微注射前,把取出的胚胎孵育在细胞培养箱的M2培养基中。
6.6、显微注射与胚胎移植
(1)准备固定针,注射针以及硅化的玻璃载玻片,往载玻片中间滴一滴被矿物油覆盖的M2培养基。
(2)使注射针依靠毛细管作用自动吸入并充满步骤2.4制备的显微注射混合液,将注射针装载至显微注射仪的固定柄上。
(3)转移50个胚胎至载玻片的M2培养基内,移动固定针靠近胚胎,使胚胎依靠负压作用被固定在固定针上。胚胎被固定后在高倍镜下找到细胞质,推动注射针的尖端穿过透明带,细胞膜,将混合液注射到胚胎的细胞质中。
(4)将注射过的胚胎细胞转移至新的M2培养基点中。重复步骤(3)和(4)直至所有的胚胎被注射完。注射完一组实验组后,将胚胎转移至新的KSOM培养基中,将胚胎放置于细胞培养箱中培养1-2小时或过夜。待所有胚胎均被注射后,排除因机械力损伤致死的胚胎,将健康的胚胎转移至新的KSOM培养基中。
(5)向假孕母鼠腹腔注射600μL阿佛丁,将假孕母鼠麻醉。使用剃毛器将母鼠背部的毛剔除。使用70%乙醇擦拭刹剃毛后的皮肤。
(6)在卵巢位置处剪开一个小口,使用钝头镊子牵拉着卵巢的脂肪垫从而将卵巢拉出,并同时用止血钳将卵巢固定于外侧,使用钝头镊子找到位于卵巢囊下侧的输卵管漏斗状口。
(7)使转移针依次吸入M2培养基,两个小气泡,约15个胚胎,气泡是为了便于观察胚胎在转移针中的位置。
(8)轻轻地剖开卵巢的囊,用辕子定位输卵管的漏斗状口,将转移针伸向卵巢的开口处,然后将转移针内的胚胎打出,轻轻地将转移针撤回。
(9)释放固定卵巢脂肪垫的止血钳,将卵巢放回原来的腔洞,用缝合线分别缝合肌肉开口处与皮肤开口处。
(10)将手术后的小鼠放置于恒温37度的保温台上,待小鼠恢复知觉后,将其转移至饲养笼具中饲养,等待胚胎发育直至分娩。一般来说,移植成功的母鼠在3周后生出小鼠。
6.7、小鼠基因组鉴定
取步骤6.6小鼠出生10-15天左右,剪其脚趾进行基因组的鉴定,具体步骤如下:
6.7.1基因组提取
①剪其脚趾装入1.5mL离心管中,每管加入500μL按蛋白酶K:组织裂解液=1:500的比例配制的脚趾消化液,55℃水浴过夜;
②取出过夜消化的脚趾,室温放置10-15分钟,充分颠倒混匀,13000rpm离心15分钟。
③每管吸出400μL上清,加入等体积氯仿,充分混匀待DNA析出后,12000rpm离心10分钟。
④每管加入提前在-20℃冰箱预冷的75%酒精200μL,轻柔地混匀,12000rpm 4℃离心5min,弃上清,在洁净工作台中晾干。
⑤根据DNA量加入50-100μL去离子超纯水,55℃溶解2小时即可作为PCR模板。
6.7.2基因组的鉴定
使用靶点DMA-sg3的引物对F/R(表11)获得含有靶点的DNA片段,先通过一代测序确认有双峰出现,后续再进行高通量深度测序得出编辑效率。根据高通量结果显示,hyA3A-BE4max处理组产生突变的10只F0中,有6只纯合的从CAA到TAA的终止无义突变(图19下图中编号为#BD03、#BD05、#BD07、#BD12、#BD15、#BD16的小鼠),而A3A-BE4max处理组F0中未发现从CAA到TAA的终止无义突变(图19上图)。与A3A-BE4max处理组相比,hyA3A-BE4max处理组小鼠经过纯合的突变中含有终止密码子TAA的Reads(高通量测序片段)数目占总Reads的比率高(图20)。
表11
6.8、DMD基因的表型鉴定
取来自5周大的野生型小鼠(空白对照)以及2.7.2鉴定的DMD基因突变小鼠,按如下方法进行免疫组织化学检测:
取小鼠的胫骨前肌,用酒精、PBS漂洗干净。将其放入铺有OTC胶的小正方体盒中,置于异戊烷烧杯,在液氮中冷冻,约30s取出,放入-20℃保存,之后进行冰冻切片。将制备好的切片用PBST洗3次/5min,对准组织位置画油圈,加入封闭液进行封闭,封闭1h后,分别用层粘连蛋白(Laminin)一抗或抗肌萎缩蛋白(Dystrophin)一抗(用1:500稀释后的一抗兔多克隆抗体(Abcam,ab11575)或一抗兔多克隆抗体(Abcam,ab15277))进行过夜孵育。用PBST洗3次/5min,然后以1:1000的抗兔稀释液二抗孵育2h,PBST洗3次/5min后,加入以1:100稀释的DAPI孵育10min,PBST洗3次/5min,滴加防猝灭剂,加盖玻片,指甲油封片,最后用荧光显微镜观察切片的荧光。
结果表明:与WT(+/+)和A3A-BE4max处理组小鼠(如#AD26)相比,只有hyA3A-BE4max处理组小鼠中引起DMA-sg3靶点序列第10位C到T纯合无义突变的6只F0代小鼠(如图21中的#BD03)中的DMD未得到蛋白表达(图21),这也证明DMD动物疾病模型构建成功。
根据荧光观察结果,同时获得hyA3A-BE4max和A3A-BE4max编辑后产生的F0代小鼠总结,如表12所示。
表12、hyA3A-BE4max和A3A-BE4max编辑后产生的F0突变结果对比
6.9、DMD突变小鼠的生殖系遗传分析(F0→F1)
取雌性纯合的具有DMD表型的#BD12(F0)与雄性野生型小鼠进行交配,获得8只纯合的F1,对出生的F1进行基因型鉴定,sanger测序结果发现,每只F1产生无义突变的Reads频率均超过96%,即这种无义突变可以稳定遗传到F1代(图22)。
6.10、脱靶检测
利用CRISPR RGEN Tools网站(http://www.rgenome.net)的Cas-OFFinder功能设计脱靶引物(表13):首先选择所测试工具的PAM的类型以及被测试的物种类型(如小鼠为Mus musculus(mm10)-Mouse),然后将所设计的sgRNA序列除去PAM部分填写在Query Sequences的方框内,选择错配碱基在3个以内,形成的DNA Bulge Size为在1以内,然后提交后获得相应的脱靶引物。
使用上述脱靶引物分别对经hyA3A-BE4max编辑的F0小鼠基因组DNA(以WT为空白对照)进行PCR后对产物进行高通量深度测序,获得脱靶效率结果(图23)。
表13、DMD-sg3脱靶位点及PCR引物
图23结果表明,与空白对照相比,融合蛋白hyA3A-BE4max及DMD-sg3对表13中15个与DMD-sg3靶点相似的位点(即脱靶位点)基本不发生C到T的编辑,即本申请的融合蛋 白hyA3A-BE4max及DMD-sg3基本不产生脱靶效应。
Claims (51)
- 一种提高基因编辑效率的融合蛋白,其特征在于,所述融合蛋白包括单链DNA结合蛋白功能域、核苷脱氨酶和核酸酶。
- 根据权利要求1所述的融合蛋白,其特征在于,所述融合蛋白的连接顺序为:所述核苷脱氨酶位于所述核酸酶的N端或C端,所述单链DNA结合蛋白功能域位于所述核苷脱氨酶和所述核酸酶的N端、C端和/或所述核苷脱氨酶和所述核酸酶之间。
- 根据权利要求2所述的融合蛋白,其特征在于,所述核苷脱氨酶位于所述核酸酶的N端。
- 根据权利要求3所述的融合蛋白,其特征在于,所述单链DNA结合蛋白功能域位于所述核苷脱氨酶和所述核酸酶之间。
- 根据权利要求1所述的融合蛋白,其特征在于,所述单链DNA结合蛋白包括序列特异性单链DNA结合蛋白、和/或非序列特异性单链DNA结合蛋白。
- 根据权利要求5所述的融合蛋白,其特征在于,所述非序列特异性单链DNA结合蛋白选自RPA70、RPA32、BRCA2、hnRNPK、PUF60和Rad51中的任一种或任几种;所述序列特异性单链DNA结合蛋白选自TEBP、Teb1和POT1中的任一种或任几种。
- 根据权利要求5所述的融合蛋白,其特征在于,所述单链DNA结合蛋白功能域包括如下四种结构域中的至少一种或如下四种结构域中具有与单链DNA结合功能的部分多肽片段及其任意组合:所述单链DNA结合蛋白的OB折叠、KH结构域、RRMS、涡状结构域。
- 根据权利要求5所述的融合蛋白,其特征在于,所述单链DNA结合蛋白功能域包括Rad51的DNA结合结构和/或RPA70的DNA结合结构域。
- 根据权利要求8所述的融合蛋白,其特征在于,所述Rad51的DNA结合结构域的氨基酸序列包括SEQ ID No.1所示序列;和/或,所述Rad51的DNA结合结构域的编码序列包括SEQ ID No.2所示序列。
- 根据权利要求8所述的融合蛋白,其特征在于,所述RPA70的DNA结合结构域的氨基酸序列包括SEQ ID No.11所示序列;和/或,所述RPA70的DNA结合结构域的编码序列包括SEQ ID No.12所示序列。
- 根据权利要求1所述的融合蛋白,其特征在于,所述脱氨酶包括胞嘧啶脱氨酶和/或腺苷脱氨酶。
- 根据权利要求12所述的融合蛋白,其特征在于,所述胞嘧啶脱氨酶包括来源于大鼠的胞嘧啶脱氨酶。
- 根据权利要求13所述的融合蛋白,其特征在于,所述来源于大鼠的胞嘧啶脱氨酶氨基酸序列包括SEQ ID No.3所示序列;和/或,所述来源于大鼠的胞嘧啶脱氨酶的编码序列包括SEQ ID No.4所示序列。
- 根据权利要求12所述的融合蛋白,其特征在于,所述胞嘧啶脱氨酶包 括来源于人的胞嘧啶脱氨酶APOBEC3A。
- 根据权利要求15所述的融合蛋白,其特征在于,所述来源于人的胞嘧啶脱氨酶APOBEC3A的氨基酸序列包括SEQ ID No.13所示序列;和/或,所述来源于人的胞嘧啶脱氨酶APOBEC3A的编码序列包括SEQ ID No.14所示序列。
- 根据权利要求12所述的融合蛋白,其特征在于,所述胞嘧啶脱氨酶包括胞嘧啶脱氨酶APOBEC3A的突变体,所述突变体是将所述胞嘧啶脱氨酶APOBEC3A第57位的天冬酰胺突变为甘氨酸。
- 根据权利要求17所述的融合蛋白,其特征在于,所述胞嘧啶脱氨酶APOBEC3A来源于人。
- 根据权利要求18所述的融合蛋白,其特征在于,所述胞嘧啶脱氨酶APOBEC3A的氨基酸序列包括SEQ ID No.13所示序列;和/或,所述胞嘧啶脱氨酶APOBEC3A的编码序列包括SEQ ID No.14所示序列。
- 根据权利要求17所述的融合蛋白,其特征在于,所述胞嘧啶脱氨酶APOBEC3A突变体的氨基酸序列包括SEQ ID No.15所示序列;和/或,所述胞嘧啶脱氨酶APOBEC3A的编码序列包括SEQ ID No.16所示序列。
- 根据权利要求1所述的融合蛋白,其特征在于,所述核酸酶选自Cas9、Cas3、Cas8a、Cas8b、Cas10d、Cse1、Csy1、Csn2、Cas4、Cas10、Csm2、Cmr5、Fok1、Cpf1中的一种或任意几种。
- 根据权利要求21所述的融合蛋白,其特征在于,所述核酸酶为Cas9。
- 根据权利要求22所述的融合蛋白,其特征在于,所述Cas9选自来源于肺炎链球菌、金黄色葡萄球菌、酿脓链球菌或嗜热链球菌的Cas9。
- 根据权利要求22所述的融合蛋白,其特征在于,所述Cas9选自Cas9突变体VQR-spCas9、VRER-spCas9、或spCas9n。
- 根据权利要求24所述的融合蛋白,其特征在于,所述spCas9n的氨基酸序列包括SEQ ID No.5所示序列;和/或,所述spCas9n的编码序列包括SEQ ID No.6所示序列。
- 根据权利要求1所述的融合蛋白,其特征在于,所述融合蛋白还包括NLS。
- 根据权利要求26所述的融合蛋白,其特征在于,所述NLS位于所述融合蛋白的至少一端。
- 根据权利要求26所述的融合蛋白,其特征在于,所述NLS的氨基酸序列包括SEQ ID No.7所示序列;和/或,所述NLS的编码序列包括SEQ ID No.8所示序列。
- 根据权利要求1所述的融合蛋白,其特征在于,所述融合蛋白还包括两拷贝以上的UGI。
- 根据权利要求29所述的融合蛋白,其特征在于,所述UGI位于所述融合蛋白的至少一端。
- 根据权利要求29所述的融合蛋白,其特征在于,所述UGI的氨基酸序列包括SEQ ID No.9所示序列;和/或,所述UGI的编码序列包括SEQ ID No.10所示序列。
- 如下A)-C)生物材料中的任一种:A)一种基因,编码权利要求1-31中任一所述融合蛋白;B)一种重组载体,含有A)所述基因;C)一种重组细胞或重组菌,含有权利要求1-31中任一所述的融合蛋白,或含有A)所述基因。
- 根据权利要求32所述的生物材料,其特征在于,所述细胞为T细胞、造血干细胞、骨髓细胞、血红细胞、或红细胞前体细胞。
- 一种对细胞内目的基因进行基因编辑的sgRNA,其特征在于,所述sgRNA的靶序列包括SEQ ID No.17-36中至少一种。
- 根据权利要求34所述的sgRNA,其特征在于,所述细胞为T细胞、造血干细胞、骨髓细胞、血红细胞、或红细胞前体细胞。
- 根据权利要求34所述的sgRNA,其特征在于,所述目的基因为HBG1和HBG2启动子区。
- 一种单碱基基因编辑系统,其特征在于,所述系统包括权利要求1-31中任一所述的融合蛋白、和/或权利要求32或33所述的生物材料、和sgRNA,所述sgRNA引导所述融合蛋白对目的细胞中的目的基因进行单碱基基因编辑。
- 根据权利要求37所述的单碱基基因编辑系统,其特征在于,所述sgRNA的靶序列包括SEQ ID No.17-36中至少一种;和/或,所述细胞为T细胞、造血干细胞、骨髓细胞、血红细胞、或红细胞前体细胞,和/或,所述目的基因为HBG1和HBG2启动子区。
- 权利要求1-31中任一所述融合蛋白、权利要求32或33所述的生物材料、权利要求34-36中任一所述的sgRNA、或权利要求37或38所述的单碱基基因编辑系统在制备基因编辑产品、疾病治疗和/或预防产品、动物模型或植物新品种中的应用。
- 根据权利要求39所述的应用,其特征在于,所述疾病为β血红蛋白病,所述β血红蛋白病包括β地中海贫血和/或镰刀型细胞贫血症。
- 一种提高单碱基基因编辑效率的方法,其特征在于,所述方法包括利用权利要求1-31中任一所述的融合蛋白和sgRNA引入细胞、对目的基因进行基因编辑的步骤,所述sgRNA引导所述融合蛋白对所述目的基因进行单碱基基因编辑。
- 根据权利要求41所述的方法,其特征在于,所述sgRNA的靶序列包括SEQ ID No.17-36中的至少一种;和/或,所述细胞为T细胞、造血干细胞、骨髓细胞、血红细胞、或红细胞前体细胞;和/或,所述目的基因为HBG1和HBG2启动子区。
- 一种疾病动物模型的构建方法,其特征在于:所述方法包括利用权利要 求1-31中任一所述的融合蛋白和sgRNA引入动物细胞、对目的基因进行基因编辑的步骤。
- 根据权利要求43所述的方法,其特征在于,所述sgRNA的靶序列包括SEQ ID No.17-36中的至少一种。
- 根据权利要求44所述的方法,其特征在于,所述sgRNA的靶序列包括SEQ ID No.36所示序列,所述目的基因包括DMD基因。
- 根据权利要求43所述的方法,其特征在于,所述动物为哺乳动物,和/或,所述细胞为胚胎细胞,和/或,所述引入的方式为载体转化、显微注射、转染、脂质转染、热休克、电穿孔、转导、基因枪、DEAE-葡聚糖介导的转移中的一种或任几种组合,和/或,所述引入使用权利要求1-31中任一所述的融合蛋白的mRNA和所述sgRNA进行。
- 根据权利要求46所述的方法,其特征在于,所述哺乳动物为大鼠或小鼠;和/或,当所述引入的方式为显微注射时,所述引入使用的权利要求1-31中任一所述的融合蛋白的mRNA的浓度为1-1000ng/μL。
- 根据权利要求47所述的方法,其特征在于,所述引入使用的权利要求1-31中任一所述融合蛋白的mRNA与所述引入使用的所述sgRNA的浓度比为1:(5-1)。
- 权利要求43-48中任一所述方法得到的动物模型在药物筛选、疾病治疗效果评价或疾病治病机理研究中的应用。
- 一种用于治疗和/或预防β血红蛋白病的产品,其特征在于,所述产品中包含:权利要求32中A)中所述基因和sgRNA的递送载体,所述sgRNA引导所述融合蛋白对目的细胞中目的基因进行单碱基基因编辑;所述目的基因为HBG1和HBG2启动子区。
- 根据权利要求50所述的产品,其特征在于,所述sgRNA的靶序列包括SEQ ID No.35所示序列;和/或,所述β血红蛋白病包括β地中海贫血和/或镰刀型细胞贫血症;和/或,所述细胞为T细胞、造血干细胞、骨髓细胞、血红细胞、或红细胞前体细胞。
- 根据权利要求50所述的产品,其特征在于,所述递送载体包括病毒载体、和/或非病毒载体;所述病毒载体包括腺相关病毒载体、腺病毒载体、慢病毒载体、逆转录病毒载体、和/或溶瘤病毒载体,所述非病毒载体包括阳离子高分子聚合物、和/或脂质体。
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