Fusion protein and polynucleotide thereof, base editor and application thereof in medicine preparation
阅读说明：本技术 一种融合蛋白及其多核苷酸、碱基编辑器及其在药物制备中的应用 (Fusion protein and polynucleotide thereof, base editor and application thereof in medicine preparation ) 是由 姚少华 于 2021-09-10 设计创作，主要内容包括：本发明公开了一种融合蛋白及其多核苷酸、碱基编辑器及其在药物制备中的应用。该融合蛋白包括saCas9核酸酶,以及插入在其第118～133位,和/或第685～702位氨基酸插入位点之间的脱氨酶；具体可以是在第125和126位氨基酸之间或者第693和694位氨基酸之间插入脱氨酶。本发明利用SaCas9/sgRNA/DNA复合物的晶体结构设计了一系列碱基编辑器,其中将胞嘧啶或腺苷脱氨酶镶嵌在SaCas9蛋白内部的不同位置,与现有N端融合胞嘧啶或腺苷脱氨酶的碱基编辑器,125位和693位融合的碱基编辑器具有近似或者更高的编辑活性以及不同的编辑窗口,极大地改善了碱基编辑器的编辑范围,为优化编辑结果提供了更多选择；本发明在基因修饰细胞治疗和基于基因编辑的基因治疗药物中具有重要价值。(The invention discloses a fusion protein and polynucleotide thereof, a base editor and application thereof in medicine preparation. The fusion protein comprises sacAS9 nuclease and deaminase inserted between the insertion sites of amino acids 118-133 and/or 685-702; specifically, a deaminase may be inserted between amino acids 125 and 126 or between amino acids 693 and 694. The invention designs a series of base editors by utilizing the crystal structure of a SaCas9/sgRNA/DNA compound, wherein cytosine or adenosine deaminase is inlaid at different positions in the SaCas9 protein, and the base editors fused with the cytosine or the adenosine deaminase at the N end and the positions 125 and 693 have approximate or higher editing activity and different editing windows, thereby greatly improving the editing range of the base editors and providing more choices for optimizing editing results; the invention has important value in gene modified cell therapy and gene therapy medicine based on gene editing.)
1. A fusion protein comprising a nuclease and a deaminase inserted between the sites of insertion of amino acids 118 to 133 and/or 685 to 702.
2. The fusion protein of claim 1, wherein the fusion protein comprises a nuclease and a deaminase inserted between amino acids 125 and 126 and/or 693 and 694.
3. The fusion protein of claim 1, wherein the nuclease is a SaCas9 protein, or a protein having at least 80% homology thereto.
4. The fusion protein of claim 3, wherein the SacAS9 protein contains a D10A mutation, the amino acid sequence of the protein is shown as SEQ ID NO.1, and the insertion site of the SacAS9 protein can be simultaneously inserted with different kinds of deaminases; the deaminase is at least one of cytosine deaminase and adenine deaminase.
5. The fusion protein of claim 4, further comprising a pyrimidine glycosidase inhibitor protein and a nuclear localization signal; the pyrimidine glycosidase inhibitor protein is fused at the C terminal of the SaCas9 protein; the nuclear localization signal is fused at the C end of the fusion protein.
6. The fusion protein of any one of claims 1-5, wherein the amino acid sequence of the fusion protein is as shown in any one of SEQ ID No.4, SEQ ID No.5, and SEQ ID No. 6.
7. A polynucleotide encoding the fusion protein of any one of claims 1 to 6.
8. A guide RNA/nuclease complex comprising at least one guide RNA and the fusion protein of any one of claims 1 to 6; the complex is capable of recognizing, binding to all or part of the target sequence and undergoing cleavage and deamination.
9. A vector comprising the polynucleotide of claim 7.
10. Use of the fusion protein of any one of claims 1 to 6 or the polynucleotide of claim 7 for drug screening and gene modification.
11. Use of the fusion protein of any one of claims 1 to 6, the polynucleotide of claim 7, the complex of claim 8 or the vector of claim 9 for the preparation of a gene therapy drug or a cell therapy drug.
12. A method for modifying the genome of a host cell, the method comprising:
providing at least one guide RNA and a fusion protein according to any one of claims 1 to 6 to a host cell comprising at least one target sequence to be modified; the fusion protein is capable of forming a complex with a guide RNA; the complex is capable of recognizing, binding to and optionally nicking, unwinding or cleaving all or part of at least one target sequence.
13. A pharmaceutical composition comprising the fusion protein of claim 6, the polynucleotide of claim 7, the complex of claim 8 or the vector of claim 9, and a pharmaceutically acceptable accessory ingredient.
14. A medicament for the treatment of β -hemoglobinopathy, comprising a sgRNA targeting the TGN 7-9WGATAR E-box/GATA binding motif in the red enhancer of BCL11A gene +58, and a base editor for the fusion protein or N-terminal fusion deaminase of claim 6 bound to the sgRNA;
the sgRNA spacer sequence is shown in SEQ ID NO. 7; or the sgRNA spacer sequence is a sequence which is added with, deleted with or replaced by one or more bases on the basis of the sequence shown in SEQ ID NO.7 and has the same function.
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a fusion protein, a polynucleotide and a base editor thereof, and application of the fusion protein in preparation of a medicament for treating beta-hemoglobinopathy.
Background
The CRISPR/Cas 9-derived base editor can accurately and efficiently convert one base pair to another (C/G → T/a or a/T → G/C) in the genomic DNA of interest with little Double Strand Break (DSB) initiation. The base editor typically consists of a Cas9 protein with single strand cleavage activity (nCas9) and cytidine or adenosine deaminase with activity on ssDNA substrates. Cas9 is directed to bind to its target DNA by a specific guide RNA (singleguide RNA) to form a protein-RNA-DNA ternary "R-loop" complex. The DNA strand synonymous with sgRNA (i.e., the non-complementary strand, NTS) is dissociated into relatively free single-stranded DNA (ssDNA), which provides a ssDNA substrate for deaminase.
In conventional base editors, cytidine or adenosine deaminase is fused directly to the N-terminus of nCas9 in such a way that the deaminase has a small and relatively fixed range of NTS editing, so that the deaminase can only switch bases within a small window range of NTS (called the editing window). For example, one of the most widely used spCas 9-derived cytosine base editors, BE3, typically catalyzes base conversion within positions 4 to 8 (NGG PAM is counted as 21-23). How to further optimize the base editor to improve the editing efficiency, or enlarge the editing window, or improve the specificity is the key to improve the efficiency of the base editor and expand the application range.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a fusion protein and a polynucleotide thereof, a base editor and application thereof in medicine preparation, and provides a fusion protein of SaCas9, which is obtained by inserting cytosine deaminase and/or adenine deaminase between the 118 th to 133 th positions and/or 685 th to 702 th positions of SaCas9, and specifically between the 125 th and 126 th positions or between the 693 th and 694 th positions of amino acid.
The invention designs a series of base editors by using the crystal structure of a SaCas9/sgRNA/DNA compound, wherein cytosine or adenosine deaminase is embedded at different positions inside a SaCas9 n. The properties of the obtained base editors were characterized and found to have different editing ranges, and the base editors fused at positions 125 and 693 each had an advantage in the effect of base editing as compared with the base editors fused at the N-terminus with cytosine or adenosine deaminase. The internal mosaic SaCas9 base editor described above greatly improves the target range, providing more options for optimization of site editing schemes of particular interest.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a fusion protein comprising a nuclease and a deaminase inserted between sites of insertion of amino acids 118 to 133 and/or 685 to 702 thereof.
Further, the fusion protein nuclease, and deaminase inserted between amino acids 125 and 126, and/or 693 and 694 thereof.
Further, the nuclease is a SaCas9 protein, or a protein having at least 80% homology thereto.
Further, the SaCas9 protein contains a D10A mutation, the amino acid sequence of the SaCas9 protein is shown in SEQ ID NO.1, and the insertion site of the SaCas9 protein can be simultaneously inserted with different kinds of deaminases.
Further, the deaminase is at least one of cytosine deaminase and adenine deaminase.
Further, cytosine deaminase contains a Y130F mutation, the amino acid sequence of which is shown in SEQ DI NO. 2.
Further, the adenine deaminase is TadA-8e, and the amino acid sequence thereof is shown in SEQ DI NO. 3.
Further, the deaminase is linked to the SaCas9 protein by a linker, which is XTEN.
Further, the fusion protein fuses 1 cytosine deaminase, and the cytosine deaminase is inserted between the 125 th and 126 th amino acids or between the 693 th and 694 th amino acids of the SacAS 9.
Further, the fusion protein fuses 1 adenine deaminase, and the adenine deaminase is inserted between the 125 th and 126 th amino acids or between the 693 th and 694 th amino acids of the SaCas 9.
Further, the fusion protein fused 1 cytosine deaminase and 1 adenine deaminase, and inserted the cytosine deaminase and adenine deaminase between amino acids 693 and 694 of said SaCas 9.
Further, adenine deaminase is fused to the N-terminus of cytosine deaminase.
Further, the fusion protein also comprises a pyrimidine glycosidase inhibitor protein and a nuclear localization signal, wherein the pyrimidine glycosidase inhibitor protein is fused at the C terminal of the SaCas9 protein; the nuclear localization signal is fused to the C-terminus of the fusion protein.
The amino acid sequence of the fusion protein is shown in any one of SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.
A polynucleotide encoding the above fusion protein.
A guide RNA/nuclease complex comprising at least one guide RNA and the above fusion protein; the complex is capable of recognizing, binding to, and optionally nicking, unwinding, or cleaving all or part of the target sequence.
A vector comprising the polynucleotide.
A host cell comprising the polynucleotide, or vector, and capable of expressing the fusion protein.
"host cell" generally refers to a biological cell. A cell may be the basic structure, function and/or biological unit of a living organism. The cells may be derived from any organism having one or more cells. Examples of host cells include, but are not limited to: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoal cells, cells from plants, animal cells, cells of mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Furthermore, the cells may be stem cells or progenitor cells.
A base editor, which comprises the fusion protein or polynucleotide and a carrier plasmid.
The fusion protein, polynucleotide or base editor can be used in drug screening and gene modification.
The fusion protein, polynucleotide, compound, vector or base editor can be used for preparing gene therapy medicines or cell therapy medicines.
A method for modifying the genome of a host cell, the method comprising: providing a host cell comprising at least one target sequence to be modified with at least one guide RNA and the above-described fusion protein or base editor; the guide RNA and the fusion protein or base editor are capable of forming a complex; the complex is capable of recognizing, binding to and optionally nicking, unwinding or cleaving all or part of at least one target sequence.
A pharmaceutical composition comprises the polynucleotide, the complex, the carrier or the base editor and pharmaceutically acceptable auxiliary components.
The fusion protein, polynucleotide, complex or base editor is used in preparing medicine for treating beta-hemoglobinopathy.
A drug for treating β -hemoglobinopathy, which comprises sgRNA targeting TGN 7-9WGATAR E-box/GATA binding motif in the BCL11A gene +58 red enhancer, and a base editor for the above fusion protein or N-terminal fusion deaminase;
the sgRNA spacer sequence is shown in SEQ ID NO. 7; or a sequence which is added with, deleted from or substituted for one or more bases on the basis of the sequence shown in SEQ ID NO.7 and has the same function.
Further, the promoter also comprises a PAM sequence for guiding the SaCas9 to recognize a target gene, and the specific sequence is AAGGGT.
Further, the deaminase is at least one of cytosine deaminase and adenine deaminase.
The SaCas9 used in the embodiment of the invention is SaCas9 mutated by D10A, and the amino acid sequence is shown as SEQ ID No. 1. A3A is Y130F mutated A3A, and the amino acid sequence is shown in SEQ ID No. 2. The TadA used is TadA-8e, the amino acid sequence is shown in SEQ ID No.3, and the linker is XTEN.
CBE fusion proteins based on SaCas 9: the amino acid sequence of SaCBE-125 is shown in SEQ ID No. 4; the amino acid sequence of SaCBE-693 is shown in SEQ ID No. 5.
CABE fusion proteins based on SaCas 9: the amino acid sequence of Sa-CABE-693 is shown in SEQ ID No. 6.
In addition, more fusion proteins can be designed, such as SaCBE-269, SaCBE-593, N-terminal fusion cytosine deaminase editor Sa-CBE-N, N-terminal fusion double-base editor Sa-CABE-N, SaABE-125, SaABE-269, SaABE-593, SaABE-693, N-terminal fusion adenine deaminase editor Sa-ABE-N, etc.
The invention has the beneficial effects that:
1. by embedding cytosine deaminase inside SaCas9, the present invention obtains a series of base editors with different window ranges. Compared with an N-terminal fused base editing tool (SaCBE-N), the SaCBE-125 reduces the editing window from 3-14 to 8-15, and the SaCBE-693 widens to 3-18; and has lower DNA off-target editing.
2. In vitro verification of the mutation site of the PKU mouse model proves that the SaCBE-125 with a narrow window in the invention provides a more effective and accurate base editing scheme for mutation of C → T in PAH mutation hotspot H263Y in Phenylketonuria (PKU). And the ratio of functional editing is further improved by optimizing the length of the sgRNA.
3. By embedding two base deaminases at the inner 693aa position of SacAS9, a dual-function base editor Sa-CABE-693 was developed. Compared with a single base editing tool, Sa-CABE-693 can correct more mutation combinations, namely, the A & C can be edited simultaneously. It exhibits a further broadening of the C-to-T window compared to the N-terminal fused double base editing tool, and slightly improved A → G editing at some targets.
4. Through editing the BCL11A gene +58 th erythroid enhancer, the double-base editing tool developed by the invention is proved, and Sa-CABE-693 is more effective than the traditional double-base editing tool in introducing mutation for treating beta-hemoglobinopathy. Therefore, Sa-CABE-693 is not only a single base editing tool, but also a base editor for simultaneously editing two bases. For gene therapy, this property provides more options for the need for genomic diversity.
Drawings
FIG. 1 is a diagram showing the construction and activity verification of Sa-CBEs of chimeric A3A in SaCas9 domain in example 1;
FIG. 2 is a diagram showing the construction and activity verification of Sa-ABEs of chimeric TadA-8e in the SaCas9 domain in example 2;
FIG. 3 is a graph showing the results of off-target detection in example 3;
FIG. 4 is a diagram of the editing and optimization of the PKU target by SaCBE-125 in example 4;
FIG. 5 is a diagram of an editing window for Sa-CABE-693 design and A & C in example 5;
FIG. 6 is a graph of sgRNA design and results for Sa-CABE-693 targeting the enhancer +58 of the BCL11A gene in example 6.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1 SaCas9 Domain chimeric A3A Sa-CBEs construction and Activity verification
1. Design and construction of deaminase-embedded CBE editor in SaCas9 domain
Through visual analysis of the crystal structure of the SacAS9/sgRNA/DNA complex (the reference PDB number is 5XAW), insertion sites are designed at the REC domain 125aa and 269aa position of the SacAS9, the HNH domain 593aa position and the RUVC domain 693aa position, the positions of the insertion sites relative to NTS are shown in figure 1a, SaCBE-125, SaCBE-269, SaCBE-593, SaCBE-693 and other SaCBEs with deaminase embedded inside are respectively constructed, the construction scheme is shown in figure 1b, XTEN is a linker sequence connecting the deaminase (A3A) and the SacAS9, UGI is uracil glycosidase inhibitory protein, and NLS is a nuclear localization signal.
2. Activity Window of deaminase-embedded CBE editor within the SaCas9 Domain
The base editors described above were transfected into 8 endogenous targets and were found to have different editing ranges compared to the N-terminal fusion deaminase editor of SaCas9 (Sa-CBE-N), as shown in FIG. 1 c. In most targets, the SacBE-125 editing window obviously has the tendency of near PAM end offset and shrinkage, the SacBE-693 editing window has the tendency of widening and has higher editing efficiency in all targets, the SacBE-593 editing window also has the tendency of widening but has lower efficiency on some targets, and the SacBE-269 editing window has no obvious change.
In order to analyze the editing window more intuitively and specifically, the invention summarizes the editing efficiency on all sgrnas and summarizes the window characteristics of the editors, as shown in fig. 1 d. Firstly, the editing efficiency of SaCBE-693 is highest, and compared with Sa-CBE-N, the window is widened from original 3-14 to 3-18, and about 3-4 bases are widened. Second, the SacBE-125 edit window is 8-15, which shrinks significantly and there is a tendency for edits at C12 and C13 to converge. In addition, while SacBE-593 also broadens the editing window (3-18), its editing activity is very low at some targets, whereas the editing window (3-14) of SacBE-269 does not undergo significant changes.
Example 2 validation of the Activity of the adenine deaminase-inserted ABE editor within the SaCas9 Domain
The above insertion of cytosine deaminase A3A into SaCas9 resulted in a series of different editing windows of Sa-CBEs. But for ABE systems it is not yet clear whether there are the same window characteristics. According to the invention, TadA-8e is embedded into the same 125aa, 269aa, 593aa and 693aa positions of SaCas9, so that the window characteristic of the Sa-ABE system is verified.
As shown in FIG. 2a, SaABE-125, SaABE-269, SaABE-593 and SaABE-693 were constructed. Next, editing activity was tested at 6 endogenous targets. As shown in fig. 2b, SaABE-125 was found to edit less C or near PAM-end bias at most targets (RUNX #14, EXMI #6, Site1, Site6) compared to the N-fused Sa-ABE-N editor, but both windows showed a consistent range at the Site11 target. The window-widening editors, SacBE-693 and SacBE-593, previously validated on CBE did not significantly broaden the editing range in the ABE system, except for broadening of 1-2 bases near the PAM end as shown on RUNX1#14, FANCF # 2. SaABE-269 still showed a consistent window range with Sa-ABE-N. Thus, although the chimeric ABE editor within the SaCas9 domain has similar window properties to the previous CBE system. Namely, compared with Sa-ABE-N, the editing window of SaABE-125 is narrower, SaABE-593 and SaABE-693 broaden the editing of 1-2 bases near PAM end in some targets, and Sa-ABE-269 shows a consistent editing window. However, overall, the ABE system of the chimeric deaminase within the SaCas9 domain does not extend the editing range as significantly as in the CBE system.
Example 3 off-target detection of deaminase-embedded CBE editor within the SaCas9 Domain
The measurement of off-target effects is an important indicator for the safety evaluation of a novel base editing tool. The R-artificial loop orthogonal method is a stable and reliable method for determining the sgRNA-independent sequence-dependent DNA off-target editing. However, the known artificial R-loop structure in this technology consists of dSaCas9 and SaCas9sgRNA to determine SpCas9 independent off-target editing. To determine sequence-independent off-target editing of the SaCas 9-derived editor, the present invention replaces the artificial R loop structure with dscas 9 and SpCas 9sgRNA to determine DNA off-target editing of the SaCas 9-derived editor (fig. 3 a).
As shown in fig. 3b, the total off-target edits of the editors described above, i.e., the average of the sum of the off-target edits of all C's on the target, were evaluated in the endogenous target HEK 4. The off-target efficiency of Sa-CBE-N is found to be about 30-40% at C3 and C5; whereas the editor embedded within the domain of the present invention shows about 30% off-target editing at C5, it greatly reduces off-target editing at C3. It can be seen that the total off-target editing of the novel base editor designed by the present invention is lower.
Example 4 application of SacBE-125 editor to PKU
SaCBE-125 with a narrower window is more beneficial to the correction of disease sites caused by single nucleotide variations, so the experiment takes SaCBE-125 as an example to verify the potential of the editor in gene therapy. The mutation of C → T in the hotspot H263Y of PAH mutation in Phenylketonuria (PKU) is a very promising therapeutic strategy known to restore phenotype and blood phenylalanine levels by base editing in mouse models. This experiment analyzed the amino acid coding for the substitution of T for C in the PKU site, as shown in FIG. 4 a. Editing of C828 results in a stop codon (nonsense mutation), editing of C836 results in an amino acid mutation of H → Y (missense mutation), and the remaining C has no effect on the coding. Previous studies by the inventors have demonstrated that this missense mutation does not affect the activity of the PAH enzyme.
To compare the editing of SaCBE-125 and Sa-CBE-N at the PKU site, we first constructed HEK293 cell line containing the F263S mutation in the PAH gene. As shown in FIG. 4b, after transfection of cells, the two edits to C835 were found to be identical (33% and 34% for Sa-CBE and Sa-CBE-125, respectively), and Sa-CBE-N edited about 10% of non-target C828, while SaCBE-125 edited C828 hardly. Thus, compared to Sa-CBE-N, the editing of PKU sites by our protocol does not result in the generation of stop codons, but instead focuses on editing the target C835, thereby more efficiently introducing beneficial mutations for the treatment of PAH in PKU
In order to further optimize the editing result of the PKU target, PKU-sgRNA with the lengths of 18bp, 19bp, 20bp and 21bp spacer sequences (spacers) is constructed in the experiment, and the influence of the change of the lengths of the spacer sequences (spacers) of the sgRNA on the editing window or the editing efficiency of an editor is tested. The results are shown in FIG. 4 c: the change of the length of the spacer sequence (spacer) obviously affects the editing efficiency of the SacBE-125, the influence of an editing window is small, the target C835 editing is further improved along with the shortening of the spacer sequence (spacer), and the non-target C828 is still not edited, so that the proportion of functional editing is improved; for Sa-CBE-N, the change of the length of the spacer sequence (spacer) not only obviously influences the editing efficiency, but also changes the editing window, and for 18bp and 19bpPKU-sgRNA, the editing of the non-target C828 is improved, so that the proportion of functional editing is reduced. Therefore, the ratio of the functional editing of the SacBE-125 to the PKU locus can be improved by optimizing the length of the sgRNA spacer sequence (spacer) to 18bp and 19 bp.
The above results show that the editor of the present invention can be applied to editing complex sites with multiple bases C in the window range, i.e., in addition to the target C, there are non-target C.
Example 5 construction and Activity verification of A & C double-base editor Sa-CABE-693
The foregoing examples show that the SacBE-693 editor developed by the present invention greatly broadens the editing window, i.e., more bases C within the window can be edited. However, this CBE system also can only edit a single base type, i.e. deaminate cytosines. To further broaden the scope of editing, the present invention further optimizes the editor to edit more base types, enabling simultaneous editing of different base types within the same spacer sequence (spacer).
Thus, two deaminases, TadA-8e and A3A, were embedded at position 693aa of the SaCas9, constructing Sa-CABE-693, as shown in FIG. 5 a. This experiment further compared the double-base editing scheme of deaminase-embedded in the SacAS9 domain (Sa-CABE-693) with the traditional N-terminal fusion double-base editing scheme (Sa-CABE-N). As shown in FIG. 5b, in endogenous targets such as HEK4-4, C5-5: compared with Sa-CABE-N, the Sa-CABE-693 of the invention has wider C → T editing range. Sa-CABE-693 slightly improved the editing ratio for A → G except in the EXM1-2 target, while the editing ratio for A → G remained consistent in almost all targets.
The results show that compared with the traditional N-terminal fusion double-base editing scheme (Sa-CABE-N), the Sa-CABE-693 disclosed by the invention has a wider C → T editing range, and the A → G conversion ratio is slightly improved on certain target points, so that more mutation combinations can be generated.
Example 6 the Sa-CABE-693 targeting the +58 enhancer of the BLC11A gene of the present invention can more effectively introduce mutations for the treatment of beta-hemoglobinopathies
This example further demonstrates the potential of Sa-CABE-693 for gene therapy. In β -haemoglobin patients, inhibition of BCL11A expression is a very promising therapeutic approach known to treat this disease. Mutation of the TGN 7-9WGATAR E-box/GATA binding motif on the red enhancer of BCL11A at position +58 resulted in decreased expression of BCL11A, thereby inducing fetal hemoglobin upregulation.
First, the present invention designed BCL11A sgRNA to target the TGN 7-9WGATAR E-box/GATA binding motif, as shown in figure 6 a. Next, the editing ability of Sa-CABE-693 and Sa-CABE-N on the BCL11A sgRNA targeting site was compared. The results are shown in FIG. 6b, which is a graphical representation of the comparison of base editing efficiency at enhancer position +58 of the BCL11A gene 72 hours after transfection of HEK293T cells with Sa-CABE-693 and Sa-CABE-N, where the abscissa is the position in the target site for different As or Cs and the ordinate is the A to G or C to T editing efficiency, and the data and error bars represent the mean and standard deviation of three independent experiments.
As can be seen from FIG. 6b, compared to Sa-CABE-N, the Sa-CABE-693 of the present invention has a wider editing window for C → T: Sa-CABE-693 can edit C17 efficiently, and Sa-CABE-N has little editing effect on C17. In addition, the A → G edit by both shows a consistent result.
The above results show that the BLC11A gene +58 enhancer targeted by the present invention can introduce mutations more effectively in the treatment of β -hemoglobinopathy than the N-terminal fusion deaminase Sa-CABE-N.
It should be appreciated that the particular features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in any one or more embodiments. Furthermore, the various embodiments and features of the various embodiments described in this specification can be combined and combined by one skilled in the art without contradiction.
Sequence listing
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Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val Gly
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Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly Val
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Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile Gln
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Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His Ser
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Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu Ser
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Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu Ala
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Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr Gly
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Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala Leu
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Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys Asp
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Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg Arg
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Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys Asp
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Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe Pro
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Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr Asn
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Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn Glu
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Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe Lys
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Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu Val
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Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys Pro
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Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr Ala
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Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala Lys
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Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu Thr
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Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln Gln
420 425 430
Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro Val
435 440 445
Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile Ile
450 455 460
Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg Glu
465 470 475 480
Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys Arg
485 490 495
Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr Gly
500 505 510
Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp Met
515 520 525
Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu Asp
530 535 540
Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro Arg
545 550 555 560
Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys Gln
565 570 575
Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu Ser
580 585 590
Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile Leu
595 600 605
Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu Tyr
610 615 620
Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp Phe
625 630 635 640
Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu Met
645 650 655
Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys Val
660 665 670
Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp Lys
675 680 685
Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp Ala
690 695 700
Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys Leu
705 710 715 720
Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys Gln
725 730 735
Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu Ile
740 745 750
Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp Tyr
755 760 765
Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Lys Leu Ile Asn
770 775 780
Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu Ile
785 790 795 800
Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu Lys
805 810 815
Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His Asp
820 825 830
Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly Asp
835 840 845
Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr Leu
850 855 860
Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile Lys
865 870 875 880
Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp Tyr
885 890 895
Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr Arg
900 905 910
Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val Lys
915 920 925
Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser Lys
930 935 940
Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala Glu
945 950 955 960
Phe Ile Ala Ser Phe Tyr Lys Asn Asp Leu Ile Lys Ile Asn Gly Glu
965 970 975
Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile Glu
980 985 990
Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met Asn
995 1000 1005
Asp Lys Arg Pro Pro His Ile Ile Lys Thr Ile Ala Ser Lys Thr Gln
1010 1015 1020
Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr Glu Val
1025 1030 1035 1040
Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys
1045 1050
<210> 2
<211> 199
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His
1 5 10 15
Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr
20 25 30
Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met
35 40 45
Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys
50 55 60
Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro
65 70 75 80
Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile
85 90 95
Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala
100 105 110
Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg
115 120 125
Ile Phe Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg
130 135 140
Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His
145 150 155 160
Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp
165 170 175
Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala
180 185 190
Ile Leu Gln Asn Gln Gly Asn
195
<210> 3
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys Arg Gly Ala
100 105 110
Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Ile Asn
165
<210> 4
<211> 1414
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val Gly
1 5 10 15
Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly Val
20 25 30
Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg Ser
35 40 45
Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile Gln
50 55 60
Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His Ser
65 70 75 80
Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu Ser
85 90 95
Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu Ala
100 105 110
Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Ser Gly Ser
115 120 125
Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Met Glu Ala
130 135 140
Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr
145 150 155 160
Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr
165 170 175
Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln His
180 185 190
Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly Phe Tyr
195 200 205
Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu Gln
210 215 220
Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser
225 230 235 240
Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe Leu Gln
245 250 255
Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg Ile Phe Asp
260 265 270
Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp Ala Gly
275 280 285
Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His Cys Trp Asp
290 295 300
Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu
305 310 315 320
Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln
325 330 335
Asn Gln Gly Asn Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala
340 345 350
Thr Pro Glu Ser Asp Thr Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile
355 360 365
Ser Arg Asn Ser Lys Ala Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln
370 375 380
Leu Glu Arg Leu Lys Lys Asp Gly Glu Val Arg Gly Ser Ile Asn Arg
385 390 395 400
Phe Lys Thr Ser Asp Tyr Val Lys Glu Ala Lys Gln Leu Leu Lys Val
405 410 415
Gln Lys Ala Tyr His Gln Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile
420 425 430
Asp Leu Leu Glu Thr Arg Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly
435 440 445
Ser Pro Phe Gly Trp Lys Asp Ile Lys Glu Trp Tyr Glu Met Leu Met
450 455 460
Gly His Cys Thr Tyr Phe Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala
465 470 475 480
Tyr Asn Ala Asp Leu Tyr Asn Ala Leu Asn Asp Leu Asn Asn Leu Val
485 490 495
Ile Thr Arg Asp Glu Asn Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln
500 505 510
Ile Ile Glu Asn Val Phe Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln
515 520 525
Ile Ala Lys Glu Ile Leu Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg
530 535 540
Val Thr Ser Thr Gly Lys Pro Glu Phe Thr Asn Leu Lys Val Tyr His
545 550 555 560
Asp Ile Lys Asp Ile Thr Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu
565 570 575
Leu Leu Asp Gln Ile Ala Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu
580 585 590
Asp Ile Gln Glu Glu Leu Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu
595 600 605
Glu Ile Glu Gln Ile Ser Asn Leu Lys Gly Tyr Thr Gly Thr His Asn
610 615 620
Leu Ser Leu Lys Ala Ile Asn Leu Ile Leu Asp Glu Leu Trp His Thr
625 630 635 640
Asn Asp Asn Gln Ile Ala Ile Phe Asn Arg Leu Lys Leu Val Pro Lys
645 650 655
Lys Val Asp Leu Ser Gln Gln Lys Glu Ile Pro Thr Thr Leu Val Asp
660 665 670
Asp Phe Ile Leu Ser Pro Val Val Lys Arg Ser Phe Ile Gln Ser Ile
675 680 685
Lys Val Ile Asn Ala Ile Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile
690 695 700
Ile Ile Glu Leu Ala Arg Glu Lys Asn Ser Lys Asp Ala Gln Lys Met
705 710 715 720
Ile Asn Glu Met Gln Lys Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu
725 730 735
Glu Ile Ile Arg Thr Thr Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu
740 745 750
Lys Ile Lys Leu His Asp Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu
755 760 765
Glu Ala Ile Pro Leu Glu Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu
770 775 780
Val Asp His Ile Ile Pro Arg Ser Val Ser Phe Asp Asn Ser Phe Asn
785 790 795 800
Asn Lys Val Leu Val Lys Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg
805 810 815
Thr Pro Phe Gln Tyr Leu Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu
820 825 830
Thr Phe Lys Lys His Ile Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile
835 840 845
Ser Lys Thr Lys Lys Glu Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg
850 855 860
Phe Ser Val Gln Lys Asp Phe Ile Asn Arg Asn Leu Val Asp Thr Arg
865 870 875 880
Tyr Ala Thr Arg Gly Leu Met Asn Leu Leu Arg Ser Tyr Phe Arg Val
885 890 895
Asn Asn Leu Asp Val Lys Val Lys Ser Ile Asn Gly Gly Phe Thr Ser
900 905 910
Phe Leu Arg Arg Lys Trp Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr
915 920 925
Lys His His Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile
930 935 940
Phe Lys Glu Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn
945 950 955 960
Gln Met Phe Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr
965 970 975
Glu Gln Glu Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His
980 985 990
Ile Lys Asp Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys
995 1000 1005
Pro Asn Arg Lys Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp
1010 1015 1020
Asp Lys Gly Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp
1025 1030 1035 1040
Lys Asp Asn Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys
1045 1050 1055
Leu Leu Met Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu
1060 1065 1070
Ile Met Glu Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr
1075 1080 1085
Glu Glu Thr Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly
1090 1095 1100
Pro Val Ile Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His
1105 1110 1115 1120
Leu Asp Ile Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys
1125 1130 1135
Leu Ser Leu Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val
1140 1145 1150
Tyr Lys Phe Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn
1155 1160 1165
Tyr Tyr Glu Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys
1170 1175 1180
Lys Ile Ser Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Lys Asn Asp
1185 1190 1195 1200
Leu Ile Lys Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn
1205 1210 1215
Asp Leu Leu Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg
1220 1225 1230
Glu Tyr Leu Glu Asn Met Asn Asp Lys Arg Pro Pro His Ile Ile Lys
1235 1240 1245
Thr Ile Ala Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile
1250 1255 1260
Leu Gly Asn Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile
1265 1270 1275 1280
Lys Lys Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser Asp Tyr
1285 1290 1295
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp
1300 1305 1310
Asp Asp Asp Lys Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu
1315 1320 1325
Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu
1330 1335 1340
Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile
1345 1350 1355 1360
Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu
1365 1370 1375
Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln
1380 1385 1390
Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Pro
1395 1400 1405
Lys Lys Lys Arg Lys Val
1410
<210> 5
<211> 1414
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val Gly
1 5 10 15
Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly Val
20 25 30
Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg Ser
35 40 45
Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile Gln
50 55 60
Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His Ser
65 70 75 80
Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu Ser
85 90 95
Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu Ala
100 105 110
Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr Gly
115 120 125
Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala Leu
130 135 140
Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys Asp
145 150 155 160
Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr Val
165 170 175
Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln Leu
180 185 190
Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg Arg
195 200 205
Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys Asp
210 215 220
Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe Pro
225 230 235 240
Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr Asn
245 250 255
Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn Glu
260 265 270
Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe Lys
275 280 285
Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu Val
290 295 300
Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys Pro
305 310 315 320
Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr Ala
325 330 335
Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala Lys
340 345 350
Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu Thr
355 360 365
Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser Asn
370 375 380
Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile Asn
385 390 395 400
Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala Ile
405 410 415
Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln Gln
420 425 430
Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro Val
435 440 445
Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile Ile
450 455 460
Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg Glu
465 470 475 480
Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys Arg
485 490 495
Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr Gly
500 505 510
Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp Met
515 520 525
Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu Asp
530 535 540
Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro Arg
545 550 555 560
Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys Gln
565 570 575
Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu Ser
580 585 590
Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile Leu
595 600 605
Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu Tyr
610 615 620
Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp Phe
625 630 635 640
Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu Met
645 650 655
Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys Val
660 665 670
Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp Lys
675 680 685
Phe Lys Lys Glu Arg Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
690 695 700
Ala Thr Pro Glu Ser Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His
705 710 715 720
Leu Met Asp Pro His Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly
725 730 735
Arg His Lys Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly
740 745 750
Thr Ser Val Lys Met Asp Gln His Arg Gly Phe Leu His Asn Gln Ala
755 760 765
Lys Asn Leu Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe
770 775 780
Leu Asp Leu Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg
785 790 795 800
Val Thr Trp Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala
805 810 815
Gly Glu Val Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg
820 825 830
Ile Phe Ala Ala Arg Ile Phe Asp Tyr Asp Pro Leu Tyr Lys Glu Ala
835 840 845
Leu Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr
850 855 860
Asp Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys
865 870 875 880
Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser
885 890 895
Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn Ser Gly Ser Glu
900 905 910
Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Asn Lys Gly Tyr
915 920 925
Lys His His Ala Glu Asp Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile
930 935 940
Phe Lys Glu Trp Lys Lys Leu Asp Lys Ala Lys Lys Val Met Glu Asn
945 950 955 960
Gln Met Phe Glu Glu Lys Gln Ala Glu Ser Met Pro Glu Ile Glu Thr
965 970 975
Glu Gln Glu Tyr Lys Glu Ile Phe Ile Thr Pro His Gln Ile Lys His
980 985 990
Ile Lys Asp Phe Lys Asp Tyr Lys Tyr Ser His Arg Val Asp Lys Lys
995 1000 1005
Pro Asn Arg Lys Leu Ile Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp
1010 1015 1020
Asp Lys Gly Asn Thr Leu Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp
1025 1030 1035 1040
Lys Asp Asn Asp Lys Leu Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys
1045 1050 1055
Leu Leu Met Tyr His His Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu
1060 1065 1070
Ile Met Glu Gln Tyr Gly Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr
1075 1080 1085
Glu Glu Thr Gly Asn Tyr Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly
1090 1095 1100
Pro Val Ile Lys Lys Ile Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His
1105 1110 1115 1120
Leu Asp Ile Thr Asp Asp Tyr Pro Asn Ser Arg Asn Lys Val Val Lys
1125 1130 1135
Leu Ser Leu Lys Pro Tyr Arg Phe Asp Val Tyr Leu Asp Asn Gly Val
1140 1145 1150
Tyr Lys Phe Val Thr Val Lys Asn Leu Asp Val Ile Lys Lys Glu Asn
1155 1160 1165
Tyr Tyr Glu Val Asn Ser Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys
1170 1175 1180
Lys Ile Ser Asn Gln Ala Glu Phe Ile Ala Ser Phe Tyr Lys Asn Asp
1185 1190 1195 1200
Leu Ile Lys Ile Asn Gly Glu Leu Tyr Arg Val Ile Gly Val Asn Asn
1205 1210 1215
Asp Leu Leu Asn Arg Ile Glu Val Asn Met Ile Asp Ile Thr Tyr Arg
1220 1225 1230
Glu Tyr Leu Glu Asn Met Asn Asp Lys Arg Pro Pro His Ile Ile Lys
1235 1240 1245
Thr Ile Ala Ser Lys Thr Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile
1250 1255 1260
Leu Gly Asn Leu Tyr Glu Val Lys Ser Lys Lys His Pro Gln Ile Ile
1265 1270 1275 1280
Lys Lys Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser Asp Tyr
1285 1290 1295
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp
1300 1305 1310
Asp Asp Asp Lys Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu
1315 1320 1325
Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu
1330 1335 1340
Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile
1345 1350 1355 1360
Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu
1365 1370 1375
Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln
1380 1385 1390
Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Pro
1395 1400 1405
Lys Lys Lys Arg Lys Val
1410
<210> 6
<211> 1596
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val Gly
1 5 10 15
Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly Val
20 25 30
Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg Ser
35 40 45
Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile Gln
50 55 60
Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His Ser
65 70 75 80
Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu Ser
85 90 95
Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu Ala
100 105 110
Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr Gly
115 120 125
Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala Leu
130 135 140
Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys Asp
145 150 155 160
Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr Val
165 170 175
Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln Leu
180 185 190
Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg Arg
195 200 205
Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys Asp
210 215 220
Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe Pro
225 230 235 240
Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr Asn
245 250 255
Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn Glu
260 265 270
Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe Lys
275 280 285
Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu Val
290 295 300
Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys Pro
305 310 315 320
Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr Ala
325 330 335
Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala Lys
340 345 350
Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu Thr
355 360 365
Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser Asn
370 375 380
Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile Asn
385 390 395 400
Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala Ile
405 410 415
Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln Gln
420 425 430
Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro Val
435 440 445
Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile Ile
450 455 460
Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg Glu
465 470 475 480
Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys Arg
485 490 495
Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr Gly
500 505 510
Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp Met
515 520 525
Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu Asp
530 535 540
Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro Arg
545 550 555 560
Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys Gln
565 570 575
Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu Ser
580 585 590
Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile Leu
595 600 605
Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu Tyr
610 615 620
Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp Phe
625 630 635 640
Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu Met
645 650 655
Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys Val
660 665 670
Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp Lys
675 680 685
Phe Lys Lys Glu Arg Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
690 695 700
Ala Thr Pro Glu Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met
705 710 715 720
Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val
725 730 735
Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly
740 745 750
Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile
755 760 765
Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile
770 775 780
Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly
785 790 795 800
Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn
805 810 815
Ser Lys Arg Gly Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro
820 825 830
Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu
835 840 845
Cys Ala Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe
850 855 860
Asn Ala Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Ser Glu Thr
865 870 875 880
Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Met Glu Ala Ser Pro
885 890 895
Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser Asn
900 905 910
Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr Glu Val
915 920 925
Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln His Arg Gly
930 935 940
Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly Arg
945 950 955 960
His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu Gln Leu Asp
965 970 975
Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys
980 985 990
Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe Leu Gln Glu Asn
995 1000 1005
Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg Ile Phe Asp Tyr Asp
1010 1015 1020
Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp Ala Gly Ala Gln
1025 1030 1035 1040
Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His Cys Trp Asp Thr Phe
1045 1050 1055
Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu
1060 1065 1070
His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln
1075 1080 1085
Gly Asn Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro
1090 1095 1100
Glu Ser Asn Lys Gly Tyr Lys His His Ala Glu Asp Ala Leu Ile Ile
1105 1110 1115 1120
Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys Leu Asp Lys Ala
1125 1130 1135
Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys Gln Ala Glu Ser
1140 1145 1150
Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu Ile Phe Ile Thr
1155 1160 1165
Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp Tyr Lys Tyr Ser
1170 1175 1180
His Arg Val Asp Lys Lys Pro Asn Arg Lys Leu Ile Asn Asp Thr Leu
1185 1190 1195 1200
Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu Ile Val Asn Asn
1205 1210 1215
Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu Lys Lys Leu Ile
1220 1225 1230
Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His Asp Pro Gln Thr
1235 1240 1245
Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly Asp Glu Lys Asn
1250 1255 1260
Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr Leu Thr Lys Tyr
1265 1270 1275 1280
Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile Lys Tyr Tyr Gly
1285 1290 1295
Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp Tyr Pro Asn Ser
1300 1305 1310
Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr Arg Phe Asp Val
1315 1320 1325
Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val Lys Asn Leu Asp
1330 1335 1340
Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser Lys Cys Tyr Glu
1345 1350 1355 1360
Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala Glu Phe Ile Ala
1365 1370 1375
Ser Phe Tyr Lys Asn Asp Leu Ile Lys Ile Asn Gly Glu Leu Tyr Arg
1380 1385 1390
Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile Glu Val Asn Met
1395 1400 1405
Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met Asn Asp Lys Arg
1410 1415 1420
Pro Pro His Ile Ile Lys Thr Ile Ala Ser Lys Thr Gln Ser Ile Lys
1425 1430 1435 1440
Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr Glu Val Lys Ser Lys
1445 1450 1455
Lys His Pro Gln Ile Ile Lys Lys Gly Gly Ser Pro Lys Lys Lys Arg
1460 1465 1470
Lys Val Ser Ser Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His
1475 1480 1485
Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys Ser Gly Gly Ser Thr Asn
1490 1495 1500
Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln
1505 1510 1515 1520
Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn
1525 1530 1535
Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr
1540 1545 1550
Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro
1555 1560 1565
Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met
1570 1575 1580
Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1585 1590 1595
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
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