CN113025638A - Double-stranded nucleic acid-based paper folding structure and preparation method and application thereof - Google Patents

Double-stranded nucleic acid-based paper folding structure and preparation method and application thereof Download PDF

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CN113025638A
CN113025638A CN202110282072.XA CN202110282072A CN113025638A CN 113025638 A CN113025638 A CN 113025638A CN 202110282072 A CN202110282072 A CN 202110282072A CN 113025638 A CN113025638 A CN 113025638A
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CN113025638B (en
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丁宝全
刘建兵
武田田
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National Center for Nanosccience and Technology China
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Abstract

The invention provides a double-stranded nucleic acid-based paper folding structure, a preparation method and application thereof, wherein the paper folding structure is formed by self-assembly of double-stranded nucleic acid and a gene editing compound; the gene editing complex comprises a gene editing protein and a gRNA, and the gRNA is complementarily combined with a recognition sequence on a double-stranded nucleic acid; the gene editing complex folds the double-stranded nucleic acid into a folded paper structure. According to the invention, through reasonable sequence analysis and structural design, the specific sequence recognition characteristic of the gene editing compound is combined on two sites on the double-stranded nucleic acid, so that the double-stranded nucleic acid is folded and compressed to form a nano structure, the problem of compressing the double-stranded nucleic acid is solved, the purpose of preparing the paper folding structure by taking the double-stranded nucleic acid as a frame is realized, and the paper folding structure has universality and provides an idea for researching folding delivery of linear double-stranded nucleic acid or plasmid.

Description

Double-stranded nucleic acid-based paper folding structure and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a double-stranded nucleic acid-based paper folding structure, and a preparation method and application thereof.
Background
In recent years, the nucleic acid nanotechnology has been developed greatly, and has achieved unusual performance particularly in the field of biotechnology tool development. Currently, scientists develop various self-assembled DNA nanostructures using Deoxyribonucleic acid (DNA) as a material, and have conducted intensive studies on their functions. The DNA origami structure has regularity, modifiability, programmability and good biocompatibility, and is widely applied to the fields of biological imaging, diagnosis, treatment and the like. The DNA origami structure is a nanostructure formed by folding a long single-stranded DNA (framework DNA) in a certain annealing procedure based on a series of short oligonucleotides (staple strands). The DNA origami structure is designed according to the base complementary pairing principle, and the core is to use a long single-stranded DNA as a framework to guide the assembly of the nano structure.
To date, DNA origami structures rely on long single-stranded framework DNA, largely limiting their widespread use. Double-stranded DNA widely existing in nature, as a biological macromolecule for storing biological information, can be transcribed to produce Ribonucleic acid (RNA) in cells and further translated into protein, and is an important tool for gene therapy. The gene therapy comprises gene silencing (antisense oligonucleotide or small interfering RNA and the like), gene editing (zinc finger nuclease, CRISPR/Cas system and the like), and gene delivery (DNA or RNA expression element and the like), and has very wide application prospect in the research related to disease treatment and pathological analysis. In the aspect of DNA paper folding technology, compared with the traditional single-stranded framework DNA, the double-stranded DNA is used as the framework to construct the paper folding structure, the self-assembly of the structure and the loading of a gene therapy tool can be simultaneously realized, and the DNA paper folding method is a very valuable design strategy. The plasmid is a circular double-stranded DNA macromolecule and can carry various genetic information. The novel DNA paper folding structure is constructed by using biological materials such as double-stranded DNA, plasmids and the like as a framework, and the limitation that the DNA paper folding technology is highly dependent on the single-stranded framework DNA can be broken through.
Therefore, the development of a method capable of folding double-stranded DNA such as plasmid into a nucleic acid origami structure is of great significance for the development of DNA origami materials.
Disclosure of Invention
Aiming at the defects and practical requirements of the prior art, the invention provides a double-stranded nucleic acid-based paper folding structure, and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a double-stranded nucleic acid-based paper folding structure formed by self-assembly of a double-stranded nucleic acid and a gene editing complex;
the gene editing complex comprises a gene editing protein and a gRNA, and the gRNA is complementarily combined with a recognition sequence on a double-stranded nucleic acid;
the gene editing complex folds the double-stranded nucleic acid into a folded paper structure.
According to the invention, the gene editing complex is a nucleic acid sequence recognition tool composed of a CRISPR/Cas gene editing system, and through reasonable sequence and structure design, the unique sequence recognition characteristic of the gene editing complex is utilized to recognize and combine two sites on double-stranded nucleic acid, so that different sites on the double-stranded nucleic acid are drawn close and then folded and compressed to form a nano structure, and the assembly of a paper folding structure taking the double-stranded nucleic acid as a framework is realized. The origami structure has advantages in universality, and is specifically characterized in that a double-stranded nucleic acid framework containing different coding information can be selected, and a corresponding gene editing compound is prepared by analyzing a sequence and extracting a recognition site, so that the nano origami structure is assembled.
Preferably, the double-stranded nucleic acid comprises a linear double-stranded nucleic acid and/or a circular double-stranded nucleic acid, preferably a linear plasmid and/or a circular plasmid.
Preferably, the double-stranded nucleic acid contains the original spacer sequence.
Preferably, the double stranded nucleic acid comprises NGG.
Preferably, the double-stranded nucleic acid comprises TTTN.
Preferably, the gene-editing proteins comprise inactivated Cas proteins (dead Cas protein, dCas protein), preferably inactivated Cas9(dead Cas9, dCas9) and inactivated Cas12a (dead Cas12a, dCas12a), the dCas9 and dCas12a are linked by a linker peptide (preferably a flexible linker peptide) to form a fusion protein, and dCas9 and dCas12a are activated by binding to the respective grnas, respectively.
Preferably, the gRNA includes a first gRNA and a second gRNA that complementarily bind recognition sequences adjacent to the original spacer sequences spaced apart within the double-stranded nucleic acid.
Preferably, the first gRNA directs dCas9 to bind to the NGG site of a double-stranded nucleic acid and the second gRNA directs dCas12a to bind to the TTTN site of a double-stranded nucleic acid.
Preferably, the double-stranded nucleic acid comprises a nucleic acid sequence shown in one of SEQ ID NO 1-4, wherein SEQ ID NO 1 is a double-stranded DNA framework L1500, SEQ ID NO 2 is a plasmid framework P3487, SEQ ID NO 3 is a plasmid framework P5949, and SEQ ID NO 4 is a plasmid framework P10227.
Preferably, the gRNA comprises a nucleic acid sequence shown in SEQ ID NO. 5-126;
5 (recognition site for double-stranded DNA framework 1500-9-1):
CAGAATACTGTGAGAACTCA；
6 (recognition site for double-stranded DNA framework 1500-9-2):
CTAACTGCGAATGTAATCAG；
SEQ ID NO:7 (recognition site for double-stranded DNA framework 1500-9-3):
ACTGTATAGATTCTCATACA；
SEQ ID NO:8 (recognition site for double stranded DNA framework 1500-9-4):
CATCGCTAACAGCGACGCGC；
SEQ ID NO:9 (recognition site for double stranded DNA framework 1500-9-5):
CACATGACGCACTTGTAACG；
10 (recognition site for double-stranded DNA framework 1500-9-6):
TCGCGCTGTGCACGAGTGTA；
11 (recognition sites 1500-9-7 in the double stranded DNA framework):
CGTGATATATTGTGCGATCA；
12 (recognition site for double-stranded DNA framework 1500-9-8):
TCAGCGAGTCATCTCGATGT；
13 (recognition site for double-stranded DNA framework 1500-9-9):
ACAGAGTTGCACGCACGCTA；
14 (recognition site for double-stranded DNA framework 1500-9-10):
GTAACTGTTCGTCGAACGAT；
15 (recognition site for double-stranded DNA framework 1500-9-11):
GTTCGAGATCTATGAATTAT；
16 (recognition site for double-stranded DNA framework 1500-9-12):
CAATCTCGCACGTACGAATC；
17 (recognition site for double-stranded DNA framework 1500-12-1):
CTCACTATTAGCACACATGCACA；
18 (recognition site for double-stranded DNA framework 1500-12-2):
GCAGCATCACGATGAGAGCTGTA；
19 (recognition site for double-stranded DNA framework 1500-12-3):
CGCACGCAGCGCTTGATATCACG；
20 (recognition site for double-stranded DNA framework 1500-12-4):
ATACTATCGATTGATCTCGAAGC；
21 (recognition site for double-stranded DNA framework 1500-12-5):
GTTACAAGAATCAGCGTGATAAT；
22 (recognition site for double-stranded DNA framework 1500-12-6):
GAGCGACTCTCGCGCAGCACGCG；
23 (recognition sites for the double-stranded DNA framework 1500-12-7):
ACGCTAGTTATACATGTACGTGC；
24 (recognition site for double-stranded DNA framework 1500-12-8):
GCTAGTTATTCATCACATACACT；
25 (recognition site for double-stranded DNA framework 1500-12-9):
CTATACACGCTTATTGTCTCGAC；
26 (recognition site for double-stranded DNA framework 1500-12-10):
CTTCAGTAGTCACGATAGTATCG；
27 (recognition site for double-stranded DNA framework 1500-12-11):
CGATACTATCGTGACTACTGAAG；
28 (recognition site for double-stranded DNA framework 1500-12-12):
CACGCGAGCATTCTAGTAGCGCG；
29 (recognition site 3487-9-1 of the plasmid P3487 framework):
AAAACCACCGCTACCAGCGG；
30 (recognition site 3487-9-2 of the plasmid P3487 framework):
ATTAGCAGAGCGAGGTATGT；
31 (recognition site 3487-9-3 of the plasmid P3487 framework):
TAGGTATCTCAGTTCGGTGT；
32 (recognition site 3487-9-4 of the plasmid P3487 framework):
GGCCTTAACGTCTCGCCCTT；
33 (recognition site 3487-9-5 of the plasmid P3487 framework):
TTGTTTATTGCAGCTTATAA；
34 (recognition site 3487-9-6 of the plasmid P3487 framework):
TGCTGTGCAGCTCCTCCACG；
35 (recognition sites 3487-9-7 of the plasmid P3487 framework):
TTCAGCTACCGCTACGAGGC；
36 (recognition site 3487-9-8 of the plasmid P3487 framework):
GCTGAAGGTCAGGGCGCCTT；
37 (recognition site 3487-9-9 of the plasmid P3487 framework):
ACCGAAGCCGCTAGCGCTAC；
38 (recognition site 3487-12-1 of the plasmid P3487 framework):
CTACTAGTGATCTGACGGTTCAC；
39 (recognition site 3487-12-2 of the plasmid P3487 framework):
GAGACTTGGAAATCCCCGTGAGT；
40 (recognition site 3487-12-3 of the plasmid P3487 framework):
CCCCCTATTGACGTCAATGACGG；
41 (recognition site 3487-12-4 of the plasmid P3487 framework):
GTCATATTGGACATGAGCCAATA；
42 (recognition site 3487-12-5 of the plasmid P3487 framework):
GACCGGGACGAGACGAACCCTGA；
43 (recognition site 3487-12-6 of the plasmid P3487 framework):
CACCGGACAGGTCGGTCTTGACA；
44 (recognition sites 3487-12-7 of the plasmid P3487 framework):
ACCTGCCCATTCGACCACCAAGC；
45 (recognition sites 3487-12-8 of the plasmid P3487 framework):
CCTGCTTGCCGAATATCATGGTG；
46 (recognition sites 3487-12-9 of the plasmid P3487 framework):
GCGAATCGGGAGCGGCGATACCG；
47 (recognition site 5949-9-1 of plasmid P5949 framework):
TTTAAAATTATGTTTTAAAA；
48 (recognition site 5949-9-2 of plasmid P5949 framework):
CAGCATAGCAAGTTTAAATA；
49 (recognition site 5949-9-3 of plasmid P5949 framework):
AACGGATCCAAGCTTGTCGA；
50 (recognition site 5949-9-4 of plasmid P5949 framework):
TTTACGGTAAACTGCCCACT；
51 (recognition site 5949-9-5 of plasmid P5949 framework):
CATCGCTATTACCATGGTCG；
52 (recognition site 5949-9-6 of plasmid P5949 framework):
CTCAGCTAATTACAGCCCGG；
53 (recognition site 5949-9-7 of plasmid P5949 framework):
ACAGATGGCTGGCAACTAGA；
54 (recognition site 5949-9-8 of plasmid P5949 framework):
GGACAGCAAGGGGGAGGATT；
55 (recognition site 5949-9-9 of plasmid P5949 framework):
CTTACGGTAAATGGCCCGCC；
56 (recognition site 5949-9-10 of plasmid P5949 framework):
ACACTTGATGTACTGCCAAG；
57 (recognition site 5949-9-11 of plasmid P5949 framework):
GAGGTCTATATAAGCAGAGC；
58 (recognition site 5949-9-12 of plasmid P5949 framework):
GGCGAGGGCGATGCCACCTA；
59 (recognition site 5949-9-13 of plasmid P5949 framework):
CATGCCCGAAGGCTACGTCC；
60 (recognition site 5949-9-14 of plasmid P5949 framework):
CAACATCCTGGGGCACAAGC；
61 (recognition site 5949-9-15 of plasmid P5949 framework):
CAGCACGGGGCCGTCGCCGA；
62 (recognition site 5949-12-1 of plasmid P5949 framework):
AACAAGCAAAACCAAATTAAGGG；
63 (recognition site 5949-12-2 of plasmid P5949 framework):
ATAGCCTGAAGAACGAGATCAGC；
64 (recognition site 5949-12-3 of plasmid P5949 framework):
ACACCGCATACGTCAAAGCAACC；
65 (recognition site 5949-12-4 of plasmid P5949 framework):
CCCGTCAAGCTCTAAATCGGGGG；
66 (recognition site 5949-12-5 of plasmid P5949 framework):
ATAGTGGACTCTTGTTCCAAACT；
67 (recognition site 5949-12-6 of plasmid P5949 framework):
CCCGAAAAGTGCCACCTGACGTC；
68 (recognition site 5949-12-7 of plasmid P5949 framework):
CGTGTCGCCCTTATTCCCTTTTT；
69 (recognition site 5949-12-8 of plasmid P5949 framework):
CGCCCCGAAGAACGTTTTCCAAT；
70 (recognition site 5949-12-9 of plasmid P5949 framework):
TGTGACTGGTGAGTACTCAACCA；
71 (recognition site 5949-12-10 of plasmid P5949 framework):
CACAACATGGGGGATCATGTAAC；
72 (recognition site 5949-12-11 of plasmid P5949 framework):
GTTCATCCATAGTTGCCTGACTC；
73 (recognition site 5949-12-12 of plasmid P5949 framework):
GTCATGAGATTATCAAAAAGGAT；
74 (recognition site 5949-12-13 of plasmid P5949 framework):
TTTGCCGGATCAAGAGCTACCAA；
75 (recognition sites 5949-12-14 of the plasmid P5949 framework):
TACCTCGCTCTGCTAATCCTGTT；
76 (recognition site 5949-12-15 of plasmid P5949 framework):
TCATAGCTCACGCTGTAGGTATC；
77 (recognition site 10227-9-1 of plasmid P10227 framework):
CATCGCTATTACCATGGTCG；
78 (recognition site 10227-9-2 of plasmid P10227 framework):
GCGCCAGGCGGGGCGGGGCG；
79 (recognition site 10227-9-3 of plasmid P10227 framework):
CCTATAAAAAGCGAAGCGCG；
80 (recognition site 10227-9-4 of plasmid P10227 framework):
TAATTACAGCCCGGAGGAGA；
81 (recognition site 10227-9-5 of plasmid P10227 framework):
CAAAGACGATGACGATAAGA；
82 (recognition site 10227-9-6 of plasmid P10227 framework):
CGGGGCCACTTCCTGATCGA；
83 (recognition sites 10227-9-7 of plasmid P10227 framework):
TCTTGCTCAGTCTGGCAGAC；
84 (recognition site 10227-9-8 of plasmid P10227 framework):
GTCCTTGCTCAGCTGCAGTT；
85 (recognition sites 10227-9-9 of plasmid P10227 framework):
CAGGGGGGCCTTGGTGATCT；
86 (recognition sites 10227-9-10 of plasmid P10227 framework):
CATTGACGGCGGAGCCAGCC；
87 (recognition sites 10227-9-11 of plasmid P10227 framework):
TTCGAGGAAGTGGTGGACAA；
88 (recognition sites 10227-9-12 of plasmid P10227 framework):
GGCGGGCTTTCTCATTCCCT；
89 (recognition sites 10227-9-13 of plasmid P10227 framework):
GATCGGTTCAACGCCTCCCT；
SEQ ID NO:90 (recognition sites 10227-9-14 of plasmid P10227 framework):
GTCGTCGAACAGGTGGGCAT；
91 (recognition sites 10227-9-15 of plasmid P10227 framework):
GTTTCTGTTGGCGAAGCCGT；
92 (recognition sites 10227-9-16 of plasmid P10227 framework):
TGTACTACCTGCAGAATGGG；
93 (recognition sites 10227-9-17 of plasmid P10227 framework):
CGACAACGTGCCCTCCGAAG；
94 (recognition sites 10227-9-18 of plasmid P10227 framework):
CCACGTGCTTTGTGATCTGC；
SEQ ID NO:95 (recognition sites 10227-9-19 of plasmid P10227 framework):
TCTCGCGCACTTTGTAAAAC；
96 (recognition sites 10227-9-20 of plasmid P10227 framework):
GAGCGAGCAGGAAATCGGCA；
97 (recognition sites 10227-9-21 of plasmid P10227 framework):
GCTGCTGGGGATCACCATCA；
98 (recognition sites 10227-9-22 of plasmid P10227 framework):
GTTTCCCTTCTGCAGTTCGC；
99 (recognition sites 10227-9-23 of plasmid P10227 framework):
TCTGCTCGATGATCTCGTCC；
100 (recognition sites 10227-9-24 of plasmid P10227 framework):
GGTGGTGTCAAAGTACTTGA；
101 (recognition sites 10227-9-25 of plasmid P10227 framework):
CCGGCGGCCACGAAAAAGGC；
102 (recognition site 10227-12-1 of plasmid P10227 framework):
CCGTAAGTTATGTAACGCGGAAC；
103 (recognition site 10227-12-2 of plasmid P10227 framework):
CGGTAAACTGCCCACTTGGCAGT；
104 (recognition site 10227-12-3 of plasmid P10227 framework):
CTACTTGGCAGTACATCTACGTA；
105 (recognition site 10227-12-4 of plasmid P10227 framework):
GGAAAGTCCCGTTGATTTTGGTG；
106 (recognition site 10227-12-5 of plasmid P10227 framework):
CGTCGCCGTCCAGCTCGACCAGG；
107 (recognition sites 10227-12-6 of plasmid P10227 framework):
CTCAGGGCGGACTGGGTGCTCAG；
108 (recognition sites 10227-12-7 of plasmid P10227 framework):
AGTGGACATCTTTATTGTTTAAT；
109 (recognition sites 10227-12-8 of plasmid P10227 framework):
CTGAAGGCTCTTTACTATTGCTT；
110 (recognition sites 10227-12-9 of plasmid P10227 framework):
TCTTGATTCCCACTTTGTGGTTC；
111 (recognition sites 10227-12-10 of plasmid P10227 framework):
CTCCTTACGCATCTGTGCGGTAT；
112 (recognition sites 10227-12-11 of plasmid P10227 framework):
ATAGTGGACTCTTGTTCCAAACT；
113 (recognition sites 10227-12-12 of plasmid P10227 framework):
CAATTTTATGGTGCACTCTCAGT；
114 (recognition sites 10227-12-13 of plasmid P10227 framework):
GGTGATGACGGTGAAAACCTCTG；
115 (recognition sites 10227-12-14 of plasmid P10227 framework):
TTTTTCTAAATACATTCAAATAT；
116 (recognition sites 10227-12-15 of plasmid P10227 framework):
GCTCACCCAGAAACGCTGGTGAA；
117 (recognition sites 10227-12-16 of plasmid P10227 framework):
CACAACATGGGGGATCATGTAAC；
118 (recognition sites 10227-12-17 of plasmid P10227 framework):
TCCGCCTCCATCCAGTCTATTAA；
119 (recognition sites 10227-12-18 of plasmid P10227 framework):
GTTCATCCATAGTTGCCTGACTC；
120 (recognition sites 10227-12-19 of plasmid P10227 framework):
GGTCATGAGATTATCAAAAAGGA；
121 (recognition sites 10227-12-20 of plasmid P10227 framework):
TTTGCCGGATCAAGAGCTACCAA；
122 (recognition sites 10227-12-21 of plasmid P10227 framework):
CCCCTGGAAGCTCCCTCGTGCGC；
123 (recognition sites 10227-12-22 of plasmid P10227 framework):
TACGGTTCCTGGCCTTTTGCTGG；
124 (recognition sites 10227-12-23 of plasmid P10227 framework):
TACGTCACGTATTTTGTACTAAT；
125 (recognition sites 10227-12-24 of plasmid P10227 framework):
CAGCATAGCTCTTAAACAGGTCT；
126 (recognition sites 10227-12-25 of plasmid P10227 framework):
TCTGCAGAATTGGCGCACGCGCT。
preferably, the gene-editing protein comprises the nucleic acid sequence set forth in SEQ ID NO: 127.
In a second aspect, the present invention provides a method for producing a paper folding structure according to the first aspect, the method comprising:
and constructing a gene editing compound, and mixing and incubating the gene editing compound with double-stranded nucleic acid to obtain the paper folding structure.
The preparation method of the paper folding structure is simple to operate and can realize large-scale production.
Preferably, the preparation method of the gene editing complex comprises the following steps:
mixing and incubating a gene editing protein, a first gRNA and a second gRNA to form a gene editing compound, wherein the first gRNA and the second gRNA are determined according to double-stranded nucleic acid, specifically, a Benchling analysis tool is used for marking an original interval sequence in the double-stranded nucleic acid, the spaced original interval sequence is selected, a sequence adjacent to the original interval sequence is recorded as an identification sequence, and the gRNA is designed according to the determined identification sequence.
Preferably, the molar ratio of the gene-editing protein to the first gRNA to the second gRNA is 1 (1-1.5): 1-1.5, preferably 1:1: 1.
Preferably, the incubation temperature is 30-37 ℃, for example, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃ or 37 ℃, preferably 37 ℃.
Preferably, the incubation time is 20-40 min, for example, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min or 40min, preferably 30 min.
Preferably, the molar ratio of the gene editing complex to the double-stranded nucleic acid is 1 (1-1.5), preferably 1: 1.
Preferably, the incubation temperature is 30-37 ℃, for example, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃ or 37 ℃, preferably 37 ℃.
Preferably, the incubation time is 20-40 min, for example, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min or 40min, preferably 30 min.
According to the invention, the nano paper folding structure with regular appearance and good dispersibility is prepared by accurately controlling the molar ratio and incubation conditions of the gene editing protein, the gRNA and the double-stranded nucleic acid, the self-assembly effect of the paper folding structure is influenced if the proportion of each component is not reasonable, for example, the gene editing protein is excessive compared with the gRNA and cannot be guided and combined to the double-stranded nucleic acid through the gRNA, so that the assembly effect is poor, and free gene editing protein exists in the obtained paper folding structure product.
In a third aspect, the invention provides the use of the origami structure of the first aspect for improving the stability of double-stranded nucleic acids.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, based on the characteristic that the gene editing compound identifies the double-stranded DNA sequence, the gene editing compound is combined on different sites on the double-stranded DNA frame, so that the folding and compressing effect on the double-stranded DNA is realized, and a nano paper folding structure is formed;
(2) the gene editing complex can identify any sequence near the original spacer sequence, has good combination effect and can realize large-scale production;
(3) the double-stranded nucleic acid framework in the paper folding structure can carry any gene, has any length, and has strong universality and simple preparation process;
(4) the paper folding structure of the invention can protect the plasmid from being degraded and has application potential in the aspect of gene therapy drug development.
Drawings
FIG. 1 is a schematic diagram of a method for preparing a double-stranded nucleic acid-based origami structure;
FIG. 2 shows the SDS-PAGE electrophoresis of the gene-edited protein, wherein lane 1 is a marker of a protein fragment of known length, and lane 2 is the purified gene-edited protein;
FIG. 3 is an atomic force microscope image of the gene editing complex, with a scale of 100 nm;
fig. 4 is a schematic of gRNA binding to L1500;
FIG. 5 is an atomic force microscope image of the origami structure of example 1, with a scale of 100 nm;
FIG. 6 is a schematic representation of the binding of gRNAs to P3487;
FIG. 7 is an atomic force microscope image of the origami structure of example 2, with a scale of 500 nm;
FIG. 8 is an atomic force microscope image of the origami structure of example 3, with a scale of 500 nm;
FIG. 9 is a schematic representation of the binding of gRNAs to P5949;
FIG. 10 is an atomic force microscope image of the origami structure of example 4, with a scale of 500 nm;
FIG. 11 is a schematic representation of the binding of a gRNA to P10227;
FIG. 12 is an atomic force microscope image of the origami structure of example 5, with a scale of 500 nm;
FIG. 13 shows the protective effect of the origami structure on the double-stranded DNA framework.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
The instruments and materials used in the examples are as follows:
equipment: gradient PCR instrument (Eppendorf, germany), microcentrifuge (ThermoFisher, usa), uv-vis spectrophotometer (shimadzu, japan), multimode scanning probe microscope (Bruker, germany), fully automated digital gel image analysis system (Tanon 2500).
Raw materials: deoxyribonucleic acid (DNA) sequences were obtained from Biotechnology engineering (Shanghai) Inc., and plasmids were obtained from Fenghui Biotechnology (Hunan) Inc.
Reagent: the buffer solutions used in the experiments included assembly reaction buffer (pH 6.5), PBS buffer (pH 7.4), SDS-PAGE electrophoresis buffer (pH 8.3), and TAE buffer (pH 8.3), wherein the composition of the assembly reaction buffer (pH 6.5) was: 2X 10-2mol·L-1Tris、15×10-2mol·L-1NaCl、1.0×10-4mol·L-1EDTA and 10X 10-2mol·L-1Magnesium acetate; the composition of the PBS buffer (pH 7.4) was: 136.9X 10-3mol·L-1NaCl、2.68×10- 3mol·L-1KCl、9.75×10-3mol·L-1Na2HPO4·H2O and 1.47X 10-3mol·L-1KH2PO4(ii) a The composition of the SDS-PAGE electrophoresis buffer (pH 8.3) was: 0.25 mol. L-1Tris、1.92mol·L-1Glycine and 1% sodium lauryl sulfate; the composition of the TAE buffer (pH 8.3) was: 4X 10-2mol·L-1Tris、2×10-2mol·L-1Acetic acid and 2.0X 10- 3mol·L-1EDTA. The reagents for each buffer were analytical grade, purchased from Sigma-Aldrich; the water used to prepare the buffer was diethyl cokenate treated sterile, enzyme-free water, purchased from beijing solibao technologies ltd.
The kit comprises: the Polymerase Chain Reaction (PCR) kit and the DNA gel recovery kit are purchased from precious bioengineering (Dalian) Co., Ltd; in vitro transcription kits were purchased from New England Biolabs; SDS-PAGE electrophoresis kits were purchased from ThermoFisher.
Example 1
In this example, a schematic diagram of a preparation method of a folded paper structure based on double-stranded nucleic acid is shown in fig. 1, and the specific steps are as follows:
(1) expression and purification of Gene editing proteins
The gene editing protein comprises two parts of an inactivated Cas9 and an inactivated Cas12a, and is prepared by the following steps:
constructing encoding genes of the inactivated Cas9 and the inactivated Cas12a into a pET 28a expression vector, and verifying the correctness of the sequences through sequencing; then transforming the expression plasmid into an escherichia coli Rosetta strain, and adding isopropyl-beta-D-thiogalactoside to induce gene editing protein expression; collecting thallus expressing gene editing protein, performing ultrasonic disruption, subjecting the supernatant to primary purification by affinity chromatography nickel column, purifying by molecular sieve gel chromatography, measuring the concentration of the purified protein (SEQ ID NO:127) by ultraviolet spectrophotometer, and performing SDS-PAGE electrophoresis detection;
as shown in FIG. 2, the SDS-PAGE electrophoresis of the gene-edited protein showed a band around the 300kDa molecular weight marker.
(2) Acquisition of guide RNA (gRNA)
Designing gRNA according to the binding site of the double-stranded nucleic acid, synthesizing a corresponding DNA template, adding the corresponding DNA template into a T7 transcription system, and incubating for 12 hours at 37 ℃ for transcription; adding DNase I to digest redundant DNA templates for 15min, then adding lithium chloride solution, and standing at-20 ℃ for 30 min; centrifuging at 4 deg.C and 13000rpm for 10min to precipitate RNA; removing supernatant, dissolving the obtained precipitate in water, and accurately quantifying by an ultraviolet spectrophotometer for later use;
(3) assembly of Gene editing complexes
Mixing and incubating the purified gene editing protein and gRNA (SEQ ID NO: 5-28) according to a molar ratio of 1:1:1, incubating for 30min at 37 ℃, assembling to form a gene editing compound, and carrying out atomic force microscope detection;
as shown in FIG. 3, the gene-editing complex is a small particle with a particle size of about 20nm and good dispersibility.
(4) Assembly of double-stranded nucleic acid-based paper folding structures
Mixing and incubating the prepared gene editing complex and a double-stranded nucleic acid L1500(SEQ ID NO:1) with the length of 1500bp according to the molar ratio of 1:1, wherein the incubation condition is that the incubation is carried out for 30min at 37 ℃, and the combination schematic diagram of the gRNA and the L1500 is shown in FIG. 4;
atomic force microscope detection is carried out on the origami structure, the result is shown in figure 5, the origami structure based on the double-stranded nucleic acid is successfully assembled, the structure is regular in appearance, the particle size is about 60nm, and the origami structure has good dispersibility.
Example 2
In this example, a paper folding structure is assembled by using a gene editing complex containing gRNAs shown in SEQ ID NO: 29-46 and linearized P3487(SEQ ID NO:2) with the length of 3487bp, the method is the same as that of example 1, and the schematic diagram of the combination of the gRNAs and the P3487 is shown in FIG. 6;
atomic force microscope detection is carried out on the origami structure, the result is shown in figure 7, the origami structure based on the double-stranded nucleic acid is successfully assembled, the structure is regular in appearance, the particle size is about 160nm, and the origami structure has good dispersibility.
Example 3
The frame DNA was P3487 which was not linearized as compared with example 2, and the other conditions were the same as in example 2.
Atomic force microscope detection is carried out on the origami structure, the result is shown in figure 8, the origami structure based on the double-stranded nucleic acid is successfully assembled, the structure is regular in appearance, the particle size is about 160nm, and the origami structure has good dispersibility.
Example 4
In this example, a paper folding structure is assembled by using a gene editing complex containing gRNAs shown in SEQ ID NO 47-76 and an annular P5949(SEQ ID NO 3) with the length of 5949bp, the method is the same as that of example 1, and the schematic diagram of the combination of the gRNAs and the P5949 is shown in FIG. 9;
atomic force microscope detection is carried out on the origami structure, the result is shown in figure 10, the origami structure based on the double-stranded nucleic acid is successfully assembled, the structure is regular in appearance, the particle size is about 250nm, and the origami structure has good dispersibility.
Example 5
In this example, a paper folding structure is assembled by using a gene editing complex containing gRNAs shown in SEQ ID NO 77-126 and a ring P10227(SEQ ID NO:4) with the length of 10227bp, the method is the same as that of example 1, and the schematic diagram of combination of the gRNAs and the P10227 is shown in FIG. 11;
atomic force microscope detection is carried out on the origami structure, the result is shown in figure 12, the origami structure based on the double-stranded nucleic acid is successfully assembled, the structure is regular in appearance, the particle size is about 412nm, and the origami structure has good dispersibility.
Example 6
From the results of examples 1 to 5, it is known that the plasmid double-stranded DNA in the paper folding structure becomes compressed tightly after being folded by the gene editing complex, forming a regular nanostructure. In this example, to further explore the protection effect of the origami structure on the double-stranded DNA framework, the origami structure of example 3 was dispersed in PBS buffer containing 10% serum, incubated in a 37 ℃ reactor for 0, 1, 2, 6, and 12 hours, and subjected to agarose gel electrophoresis in TAE buffer (pH 8.3).
The results are shown in fig. 13, the plasmid without the compressed folding of the gene editing complex is degraded into fragments with wide molecular weight distribution after 1h incubation, further degraded into short nucleic acid fragments after 6h incubation, and completely degraded after 12h incubation; the plasmid with the paper folding structure is not obviously degraded after being incubated for 12 hours, which indicates that the paper folding structure can block the contact of the plasmid and nuclease, thereby protecting the plasmid from being degraded.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> national center for Nano science
<120> double-stranded nucleic acid-based paper folding structure and preparation method and application thereof
<130> 20210315
<160> 127
<170> PatentIn version 3.3
<210> 1
<211> 1510
<212> DNA
<213> Artificial Synthesis
<400> 1
ttacgccgaa gcagaatact gtgagaactc aaggtcagta gagtcatccg ctgattacat 60
tcgcagttag tataggtcag tagagtcatc cattcactgt atagattctc atacatggtc 120
agtagagtca tcctgttatc actcgctgag catagaatgg tcagtagagt catcctcact 180
cagagtggaa gaagagaata ggtcagtaga gtcatcctgc gcgtcgctgt tagcgatgca 240
gtggtcagta gagtcatcct gcgcacatga cgcacttgta acgcggtcag tagagtcatc 300
cctacactcg tgcacagcgc gatcacggtc agtagagtca tccacgtaat atgagctgag 360
ctactcgtgg tcagtagagt catccagcga cagcgatcat ctgaacgtat ggtcagtaga 420
gtcatccagc tcgtgatata ttgtgcgatc atggtcagta gagtcatcct acatcgagat 480
gactcgctga tgtaggtcag tagagtcatc cggcaacaga gttgcacgca cgctatggtc 540
agtagagtca tccttatagc tacgctcaag tgagctgtgg tcagtagagt catccacgtg 600
attagagatg cgctgtatac ggtcagtaga gtcatccgat cgttcgacga acagttacgc 660
gcggtcagta gagtcatcct cgtgttcgag atctatgaat tattggtcag tagagtcatc 720
cggattcgta cgtgcgagat tgtcttggtc agtagagtcg tttacgctct cgcatataat 780
cgcgagttaa acagtagagt cgtttacacg cgagcattct agtagcgcgt aaacagtaga 840
gtcgtttagc attagtgagt gacaatgcta ttaaacagta gagtcgttta cttcagtagt 900
cacgatagta tcgtaaacag tagagtcgtt tagtgatagt taacgctgta tgtgctaaac 960
agtagagtcg tttagtcgag acaataagcg tgtatagtaa acagtagagt cgtttagcta 1020
gttattcatc acatacactt aaacagtaga gtcgtttagc acgtacatgt ataactagcg 1080
ttaaacagta gagtcgttta cgctataata gtgcgttgcc gtctaaacag tagagtcgtt 1140
tagagcgact ctcgcgcagc acgcgtaaac agtagagtcg tttaattatc acgctgattc 1200
ttgtaactaa acagtagagt cgtttaatac tatcgattga tctcgaagct aaacagtaga 1260
gtcgtttagt tcagatgaat cgatattgaa ctaaacagta gagtcgttta cgtgatatca 1320
agcgctgcgt gcgtaaacag tagagtcgtt tagcagcatc acgatgagag ctgtataaac 1380
agtagagtcg tttatgtgca tgtgtgctaa tagtgagtaa acagtagagt cgtttaagag 1440
cgagtgttca gcttcatgtt aaacagtaga gtcgtttaac gatgctatag aactcaagca 1500
gtaaacgctt 1510
<210> 2
<211> 3487
<212> DNA
<213> Artificial Synthesis
<400> 2
tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 60
cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 120
ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 180
gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 240
ttatgcccag tacatgacct tacgggactt tcctacttgg cagtacatct acgtattagt 300
catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg gatagcggtt 360
tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 420
ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga cgcaaatggg 480
cggtaggcgt gtacggtggg aggtctatat aagcagaggt cgtttagtga accgtcagat 540
cactagtagc tttattgcgg tagtttatca cagttaaatt gctaacgcag tcagtgctcg 600
actgatcaca ggtaagtatc aaggttacaa gacaggttta aggaggccaa tagaaactgg 660
gcttgtcgag acagagaaga ttcttgcgtt tctgataggc acctattggt cttactgaca 720
tccactttgc ctttctctcc acaggggtac cgaagccgct agcgctaccg gtcgccacca 780
tgcccgccat gaagatcgag tgccgcatca ccggcaccct gaacggcgtg gagttcgagc 840
tggtgggcgg cggagagggc acccccgagc agggccgcat gaccaacaag atgaagagca 900
ccaaaggcgc cctgaccttc agcccctacc tgctgagcca cgtgatgggc tacggcttct 960
accacttcgg cacctacccc agcggctacg agaacccctt cctgcacgcc atcaacaacg 1020
gcggctacac caacacccgc atcgagaagt acgaggacgg cggcgtgctg cacgtgagct 1080
tcagctaccg ctacgaggcc ggccgcgtga tcggcgactt caaggtggtg ggcaccggct 1140
tccccgagga cagcgtgatc ttcaccgaca agatcatccg cagcaacgcc accgtggagc 1200
acctgcaccc catgggcgat aacgtgctgg tgggcagctt cgcccgcacc ttcagcctgc 1260
gcgacggcgg ctactacagc ttcgtggtgg acagccacat gcacttcaag agcgccatcc 1320
accccagcat cctgcagaac gggggcccca tgttcgcctt ccgccgcgtg gaggagctgc 1380
acagcaacac cgagctgggc atcgtggagt accagcacgc cttcaagacc cccatcgcct 1440
tcgccagatc tcgagctcga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 1500
tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat 1560
aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg 1620
gaggtttttt aaagcaagta aaacctctac aaatgtggta cttaagaggg ggagaccaaa 1680
gggcgagacg ttaaggcctc acgtgacatg tgagcaaaag gccagcaaaa ggccaggaac 1740
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 1800
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 1860
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct tacgggatac 1920
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1980
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 2040
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 2100
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 2160
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 2220
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 2280
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 2340
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 2400
gaaaactcac gttaagggat tttggtcatg ccgtctcaga agaactcgtc aagaaggcga 2460
tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca 2520
gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag 2580
cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc 2640
atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg 2700
ctcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga 2760
tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctctctcgat gcgatgtttc 2820
gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca 2880
gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc 2940
acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag tacagctgcg 3000
caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcttg cagttcattc 3060
agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg 3120
aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc 3180
tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat aatattattg 3240
aagcatttat cagggttcgt ctcgtcccgg tctcctccca tgcatgtcaa tattggccat 3300
tagccatatt attcattggt tatatagcat aaatcaatat tggctattgg ccattgcata 3360
cgttgtatct atatcataat atgtacattt atattggctc atgtccaata tgaccgccat 3420
gttggcattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 3480
gcccata 3487
<210> 3
<211> 5947
<212> DNA
<213> Artificial Synthesis
<400> 3
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt taagagctat gctggaaaca gcatagcaag 300
tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg 360
ttttagagct agaaatagca agttaaaata aggctagtcc gtagcgcgtg cgccaattct 420
gcagacaaat ggctctagag ttaacggatc caagcttgtc gacggtaccc gttacataac 480
ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 540
tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 600
atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 660
ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 720
gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg 780
tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 840
atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg 900
cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg 960
cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 1020
ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc 1080
gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 1140
ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg 1200
taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg 1260
cctgaaatca ctttttttca ggttggaccg gtgaattcct agagctcgct gatcagcctc 1320
gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac 1380
cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 1440
tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 1500
ttgggaagag aatagcaggc atgctgggga gcggccgcta gttattaata gtaatcaatt 1560
acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 1620
ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 1680
cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 1740
actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 1800
aatgacggta aatggcccgc tggcattatg cccagtacat gaccttatgg gactttccta 1860
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 1920
acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 1980
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 2040
actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 2100
gagctggttt agtgaaccgt cagatccgct agcatggtga gcaagggcga ggagctgttc 2160
accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 2220
gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc 2280
accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg 2340
cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 2400
cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 2460
cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 2520
gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac 2580
aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc 2640
cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 2700
ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc 2760
aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 2820
atcactctcg gcatggacga gctgtacaag taaggaccgg gatgcagaaa ttgatgatct 2880
attaaacaat aaagatgtcc actaaaatgg aagtttttcc tgtcatactt tgttaagaag 2940
ggtgagaaca gagtacctac attttgaatg gaaggattgg agctacgggg gtgggggtgg 3000
ggtgggatta gataaatgcc tgctctttac tgaaggctct ttactattgc tttatgataa 3060
tgtttcatag ttggatatca taatttaaac aagcaaaacc aaattaaggg ccagctcatt 3120
cctcccactc atgatctata gatctataga tctctcgtgg gatcattgtt tttctcttga 3180
ttcccacttt gtggttctaa gtactgtggt ttccaaatgt gtcagtttca tagcctgaag 3240
aacgagatca gcagcctctg ttccacatac acttcattct cagtattgtt ttgccaagtt 3300
ctaattccat cagaagctgg tcgacctgca ggggcgcctg atgcggtatt ttctccttac 3360
gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 3420
ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 3480
gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 3540
ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 3600
ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 3660
acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 3720
actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg 3780
atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 3840
aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 3900
tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 3960
ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 4020
ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 4080
taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 4140
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 4200
agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 4260
catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 4320
ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 4380
atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 4440
ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 4500
gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 4560
ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 4620
ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 4680
gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 4740
ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 4800
gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 4860
ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 4920
gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt 4980
gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 5040
caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 5100
cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 5160
ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 5220
taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 5280
tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 5340
gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 5400
agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 5460
aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 5520
gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 5580
gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 5640
tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 5700
agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 5760
cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 5820
gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 5880
gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtc tcgaggctag 5940
cacgcgt 5947
<210> 4
<211> 10227
<212> DNA
<213> Artificial Synthesis
<400> 4
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt taagagctat gctggaaaca gcatagcaag 300
tttaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg 360
ttttagagct agaaatagca agttaaaata aggctagtcc gtagcgcgtg cgccaattct 420
gcagacaaat ggctctagag ttaacggatc caagcttgtc gacggtaccc gttacataac 480
ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 540
tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 600
atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 660
ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 720
gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg 780
tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 840
atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg 900
cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg 960
cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 1020
ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc 1080
gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 1140
ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg 1200
taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg 1260
cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg 1320
gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga 1380
agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc 1440
tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca 1500
gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg 1560
gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca 1620
gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg 1680
agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag 1740
aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc 1800
acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg 1860
ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc 1920
tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg 1980
tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca 2040
aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc 2100
tgcccggcga gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga 2160
cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg 2220
acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc 2280
tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga 2340
acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc 2400
accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag 2460
agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc 2520
aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac 2580
tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca 2640
gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt 2700
tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc 2760
cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga 2820
gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc 2880
agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc 2940
ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat 3000
acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg 3060
tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact 3120
tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg 3180
cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca 3240
atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca 3300
gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga 3360
agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg 3420
gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg 3480
ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc 3540
agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg 3600
gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga 3660
aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga 3720
ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca 3780
aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg 3840
agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg 3900
acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg 3960
acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca 4020
acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg 4080
ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga 4140
gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa 4200
agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc 4260
tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg 4320
atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc 4380
tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg 4440
tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa 4500
tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg 4560
agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa 4620
ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca 4680
tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt 4740
ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta 4800
agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag 4860
tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca 4920
tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag 4980
aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg 5040
gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc 5100
cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc 5160
ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga 5220
tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca 5280
aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata 5340
tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca 5400
ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc 5460
accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca 5520
aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag 5580
agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 5640
ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 5700
ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 5760
ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgctagt 5820
tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 5880
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 5940
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 6000
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 6060
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 6120
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 6180
gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 6240
ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 6300
tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 6360
tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcatggtgag 6420
caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 6480
aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 6540
gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 6600
caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 6660
cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 6720
cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 6780
catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 6840
gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 6900
ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 6960
ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 7020
cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 7080
gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaggatccgg 7140
gatgcagaaa ttgatgatct attaaacaat aaagatgtcc actaaaatgg aagtttttcc 7200
tgtcatactt tgttaagaag ggtgagaaca gagtacctac attttgaatg gaaggattgg 7260
agctacgggg gtgggggtgg ggtgggatta gataaatgcc tgctctttac tgaaggctct 7320
ttactattgc tttatgataa tgtttcatag ttggatatca taatttaaac aagcaaaacc 7380
aaattaaggg ccagctcatt cctcccactc atgatctata gatctataga tctctcgtgg 7440
gatcattgtt tttctcttga ttcccacttt gtggttctaa gtactgtggt ttccaaatgt 7500
gtcagtttca tagcctgaag aacgagatca gcagcctctg ttccacatac acttcattct 7560
cagtattgtt ttgccaagtt ctaattccat cagaagctgg tcgacctgca ggggcgcctg 7620
atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac 7680
catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 7740
tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 7800
tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 7860
gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta 7920
gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 7980
atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg 8040
atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 8100
aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta 8160
caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 8220
cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 8280
ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc 8340
tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag 8400
gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 8460
caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 8520
ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 8580
gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 8640
tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 8700
ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 8760
tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 8820
atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 8880
gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 8940
caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 9000
ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 9060
ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 9120
ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 9180
ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 9240
gtggaagccg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 9300
ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 9360
taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 9420
agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 9480
atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 9540
aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 9600
caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 9660
ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 9720
cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 9780
tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 9840
gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 9900
ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 9960
gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 10020
caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 10080
ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 10140
tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 10200
ctcacatgtc tcgaggctag cacgcgt 10227
<210> 5
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 5
cagaatactg tgagaactca 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 6
ctaactgcga atgtaatcag 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 7
actgtataga ttctcataca 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 8
catcgctaac agcgacgcgc 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 9
cacatgacgc acttgtaacg 20
<210> 10
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 10
tcgcgctgtg cacgagtgta 20
<210> 11
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 11
cgtgatatat tgtgcgatca 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 12
tcagcgagtc atctcgatgt 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 13
acagagttgc acgcacgcta 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 14
gtaactgttc gtcgaacgat 20
<210> 15
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 15
gttcgagatc tatgaattat 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 16
caatctcgca cgtacgaatc 20
<210> 17
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 17
ctcactatta gcacacatgc aca 23
<210> 18
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 18
gcagcatcac gatgagagct gta 23
<210> 19
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 19
cgcacgcagc gcttgatatc acg 23
<210> 20
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 20
atactatcga ttgatctcga agc 23
<210> 21
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 21
gttacaagaa tcagcgtgat aat 23
<210> 22
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 22
gagcgactct cgcgcagcac gcg 23
<210> 23
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 23
acgctagtta tacatgtacg tgc 23
<210> 24
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 24
gctagttatt catcacatac act 23
<210> 25
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 25
ctatacacgc ttattgtctc gac 23
<210> 26
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 26
cttcagtagt cacgatagta tcg 23
<210> 27
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 27
cgatactatc gtgactactg aag 23
<210> 28
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 28
cacgcgagca ttctagtagc gcg 23
<210> 29
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 29
aaaaccaccg ctaccagcgg 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 30
attagcagag cgaggtatgt 20
<210> 31
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 31
taggtatctc agttcggtgt 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 32
ggccttaacg tctcgccctt 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 33
ttgtttattg cagcttataa 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 34
tgctgtgcag ctcctccacg 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 35
ttcagctacc gctacgaggc 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 36
gctgaaggtc agggcgcctt 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 37
accgaagccg ctagcgctac 20
<210> 38
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 38
ctactagtga tctgacggtt cac 23
<210> 39
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 39
gagacttgga aatccccgtg agt 23
<210> 40
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 40
ccccctattg acgtcaatga cgg 23
<210> 41
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 41
gtcatattgg acatgagcca ata 23
<210> 42
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 42
gaccgggacg agacgaaccc tga 23
<210> 43
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 43
caccggacag gtcggtcttg aca 23
<210> 44
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 44
acctgcccat tcgaccacca agc 23
<210> 45
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 45
cctgcttgcc gaatatcatg gtg 23
<210> 46
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 46
gcgaatcggg agcggcgata ccg 23
<210> 47
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 47
tttaaaatta tgttttaaaa 20
<210> 48
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 48
cagcatagca agtttaaata 20
<210> 49
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 49
aacggatcca agcttgtcga 20
<210> 50
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 50
tttacggtaa actgcccact 20
<210> 51
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 51
catcgctatt accatggtcg 20
<210> 52
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 52
ctcagctaat tacagcccgg 20
<210> 53
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 53
acagatggct ggcaactaga 20
<210> 54
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 54
ggacagcaag ggggaggatt 20
<210> 55
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 55
cttacggtaa atggcccgcc 20
<210> 56
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 56
acacttgatg tactgccaag 20
<210> 57
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 57
gaggtctata taagcagagc 20
<210> 58
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 58
ggcgagggcg atgccaccta 20
<210> 59
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 59
catgcccgaa ggctacgtcc 20
<210> 60
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 60
caacatcctg gggcacaagc 20
<210> 61
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 61
cagcacgggg ccgtcgccga 20
<210> 62
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 62
aacaagcaaa accaaattaa ggg 23
<210> 63
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 63
atagcctgaa gaacgagatc agc 23
<210> 64
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 64
acaccgcata cgtcaaagca acc 23
<210> 65
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 65
cccgtcaagc tctaaatcgg ggg 23
<210> 66
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 66
atagtggact cttgttccaa act 23
<210> 67
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 67
cccgaaaagt gccacctgac gtc 23
<210> 68
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 68
cgtgtcgccc ttattccctt ttt 23
<210> 69
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 69
cgccccgaag aacgttttcc aat 23
<210> 70
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 70
tgtgactggt gagtactcaa cca 23
<210> 71
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 71
cacaacatgg gggatcatgt aac 23
<210> 72
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 72
gttcatccat agttgcctga ctc 23
<210> 73
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 73
gtcatgagat tatcaaaaag gat 23
<210> 74
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 74
tttgccggat caagagctac caa 23
<210> 75
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 75
tacctcgctc tgctaatcct gtt 23
<210> 76
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 76
tcatagctca cgctgtaggt atc 23
<210> 77
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 77
catcgctatt accatggtcg 20
<210> 78
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 78
gcgccaggcg gggcggggcg 20
<210> 79
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 79
cctataaaaa gcgaagcgcg 20
<210> 80
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 80
taattacagc ccggaggaga 20
<210> 81
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 81
caaagacgat gacgataaga 20
<210> 82
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 82
cggggccact tcctgatcga 20
<210> 83
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 83
tcttgctcag tctggcagac 20
<210> 84
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 84
gtccttgctc agctgcagtt 20
<210> 85
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 85
caggggggcc ttggtgatct 20
<210> 86
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 86
cattgacggc ggagccagcc 20
<210> 87
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 87
ttcgaggaag tggtggacaa 20
<210> 88
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 88
ggcgggcttt ctcattccct 20
<210> 89
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 89
gatcggttca acgcctccct 20
<210> 90
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 90
gtcgtcgaac aggtgggcat 20
<210> 91
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 91
gtttctgttg gcgaagccgt 20
<210> 92
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 92
tgtactacct gcagaatggg 20
<210> 93
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 93
cgacaacgtg ccctccgaag 20
<210> 94
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 94
ccacgtgctt tgtgatctgc 20
<210> 95
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 95
tctcgcgcac tttgtaaaac 20
<210> 96
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 96
gagcgagcag gaaatcggca 20
<210> 97
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 97
gctgctgggg atcaccatca 20
<210> 98
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 98
gtttcccttc tgcagttcgc 20
<210> 99
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 99
tctgctcgat gatctcgtcc 20
<210> 100
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 100
ggtggtgtca aagtacttga 20
<210> 101
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 101
ccggcggcca cgaaaaaggc 20
<210> 102
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 102
ccgtaagtta tgtaacgcgg aac 23
<210> 103
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 103
cggtaaactg cccacttggc agt 23
<210> 104
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 104
ctacttggca gtacatctac gta 23
<210> 105
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 105
ggaaagtccc gttgattttg gtg 23
<210> 106
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 106
cgtcgccgtc cagctcgacc agg 23
<210> 107
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 107
ctcagggcgg actgggtgct cag 23
<210> 108
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 108
agtggacatc tttattgttt aat 23
<210> 109
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 109
ctgaaggctc tttactattg ctt 23
<210> 110
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 110
tcttgattcc cactttgtgg ttc 23
<210> 111
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 111
ctccttacgc atctgtgcgg tat 23
<210> 112
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 112
atagtggact cttgttccaa act 23
<210> 113
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 113
caattttatg gtgcactctc agt 23
<210> 114
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 114
ggtgatgacg gtgaaaacct ctg 23
<210> 115
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 115
tttttctaaa tacattcaaa tat 23
<210> 116
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 116
gctcacccag aaacgctggt gaa 23
<210> 117
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 117
cacaacatgg gggatcatgt aac 23
<210> 118
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 118
tccgcctcca tccagtctat taa 23
<210> 119
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 119
gttcatccat agttgcctga ctc 23
<210> 120
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 120
ggtcatgaga ttatcaaaaa gga 23
<210> 121
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 121
tttgccggat caagagctac caa 23
<210> 122
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 122
cccctggaag ctccctcgtg cgc 23
<210> 123
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 123
tacggttcct ggccttttgc tgg 23
<210> 124
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 124
tacgtcacgt attttgtact aat 23
<210> 125
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 125
cagcatagct cttaaacagg tct 23
<210> 126
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 126
tctgcagaat tggcgcacgc gct 23
<210> 127
<211> 8052
<212> DNA
<213> Artificial Synthesis
<400> 127
gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040
ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggacgccatc 2520
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760
gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120
atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180
ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240
accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300
acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360
agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420
tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480
gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540
ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600
tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660
aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720
tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780
cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840
ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900
atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960
gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020
gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080
ctgtctcagc tgggaggcga cggtggcggc ggtagcggtg gcggtggcag catgacacag 4140
ttcgagggct ttaccaacct gtatcaggtg agcaagacac tgcggtttga gctgatccca 4200
cagggcaaga ccctgaagca catccaggag cagggcttca tcgaggagga caaggcccgc 4260
aatgatcact acaaggagct gaagcccatc atcgatcgga tctacaagac ctatgccgac 4320
cagtgcctgc agctggtgca gctggattgg gagaacctga gcgccgccat cgactcctat 4380
agaaaggaga aaaccgagga gacaaggaac gccctgatcg aggagcaggc cacatatcgc 4440
aatgccatcc acgactactt catcggccgg acagacaacc tgaccgatgc catcaataag 4500
agacacgccg agatctacaa gggcctgttc aaggccgagc tgtttaatgg caaggtgctg 4560
aagcagctgg gcaccgtgac cacaaccgag cacgagaacg ccctgctgcg gagcttcgac 4620
aagtttacaa cctacttctc cggcttttat gagaacagga agaacgtgtt cagcgccgag 4680
gatatcagca cagccatccc acaccgcatc gtgcaggaca acttccccaa gtttaaggag 4740
aattgtcaca tcttcacacg cctgatcacc gccgtgccca gcctgcggga gcactttgag 4800
aacgtgaaga aggccatcgg catcttcgtg agcacctcca tcgaggaggt gttttccttc 4860
cctttttata accagctgct gacacagacc cagatcgacc tgtataacca gctgctggga 4920
ggaatctctc gggaggcagg caccgagaag atcaagggcc tgaacgaggt gctgaatctg 4980
gccatccaga agaatgatga gacagcccac atcatcgcct ccctgccaca cagattcatc 5040
cccctgttta agcagatcct gtccgatagg aacaccctgt ctttcatcct ggaggagttt 5100
aagagcgacg aggaagtgat ccagtccttc tgcaagtaca agacactgct gagaaacgag 5160
aacgtgctgg agacagccga ggccctgttt aacgagctga acagcatcga cctgacacac 5220
atcttcatca gccacaagaa gctggagaca atcagcagcg ccctgtgcga ccactgggat 5280
acactgagga atgccctgta tgagcggaga atctccgagc tgacaggcaa gatcaccaag 5340
tctgccaagg agaaggtgca gcgcagcctg aagcacgagg atatcaacct gcaggagatc 5400
atctctgccg caggcaagga gctgagcgag gccttcaagc agaaaaccag cgagatcctg 5460
tcccacgcac acgccgccct ggatcagcca ctgcctacaa ccctgaagaa gcaggaggag 5520
aaggagatcc tgaagtctca gctggacagc ctgctgggcc tgtaccacct gctggactgg 5580
tttgccgtgg atgagtccaa cgaggtggac cccgagttct ctgcccggct gaccggcatc 5640
aagctggaga tggagccttc tctgagcttc tacaacaagg ccagaaatta tgccaccaag 5700
aagccctact ccgtggagaa gttcaagctg aactttcaga tgcctacact ggcctctggc 5760
tgggacgtga ataaggagaa gaacaatggc gccatcctgt ttgtgaagaa cggcctgtac 5820
tatctgggca tcatgccaaa gcagaagggc aggtataagg ccctgagctt cgagcccaca 5880
gagaaaacca gcgagggctt tgataagatg tactatgact acttccctga tgccgccaag 5940
atgatcccaa agtgcagcac ccagctgaag gccgtgacag cccactttca gacccacaca 6000
acccccatcc tgctgtccaa caatttcatc gagcctctgg agatcacaaa ggagatctac 6060
gacctgaaca atcctgagaa ggagccaaag aagtttcaga cagcctacgc caagaaaacc 6120
ggcgaccaga agggctacag agaggccctg tgcaagtgga tcgacttcac aagggatttt 6180
ctgtccaagt ataccaagac aacctctatc gatctgtcta gcctgcggcc atcctctcag 6240
tataaggacc tgggcgagta ctatgccgag ctgaatcccc tgctgtacca catcagcttc 6300
cagagaatcg ccgagaagga gatcatggat gccgtggaga caggcaagct gtacctgttc 6360
cagatctata acaaggactt tgccaagggc caccacggca agcctaatct gcacacactg 6420
tattggaccg gcctgttttc tccagagaac ctggccaaga caagcatcaa gctgaatggc 6480
caggccgagc tgttctaccg ccctaagtcc aggatgaaga ggatggcaca ccggctggga 6540
gagaagatgc tgaacaagaa gctgaaggat cagaaaaccc caatccccga caccctgtac 6600
caggagctgt acgactatgt gaatcacaga ctgtcccacg acctgtctga tgaggccagg 6660
gccctgctgc ccaacgtgat caccaaggag gtgtctcacg agatcatcaa ggataggcgc 6720
tttaccagcg acaagttctt tttccacgtg cctatcacac tgaactatca ggccgccaat 6780
tccccatcta agttcaacca gagggtgaat gcctacctga aggagcaccc cgagacacct 6840
atcatcggca tcgatcgggg cgagagaaac ctgatctata tcacagtgat cgactccacc 6900
ggcaagatcc tggagcagcg gagcctgaac accatccagc agtttgatta ccagaagaag 6960
ctggacaaca gggagaagga gagggtggca gcaaggcagg cctggtctgt ggtgggcaca 7020
atcaaggatc tgaagcaggg ctatctgagc caggtcatcc acgagatcgt ggacctgatg 7080
atccactacc aggccgtggt ggtgctggag aacctgaatt tcggctttaa gagcaagagg 7140
accggcatcg ccgagaaggc cgtgtaccag cagttcgaga agatgctgat cgataagctg 7200
aattgcctgg tgctgaagga ctatccagca gagaaagtgg gaggcgtgct gaacccatac 7260
cagctgacag accagttcac ctcctttgcc aagatgggca cccagtctgg cttcctgttt 7320
tacgtgcctg ccccatatac atctaagatc gatcccctga ccggcttcgt ggaccccttc 7380
gtgtggaaaa ccatcaagaa tcacgagagc cgcaagcact tcctggaggg cttcgacttt 7440
ctgcactacg acgtgaaaac cggcgacttc atcctgcact ttaagatgaa cagaaatctg 7500
tccttccaga ggggcctgcc cggctttatg cctgcatggg atatcgtgtt cgagaagaac 7560
gagacacagt ttgacgccaa gggcacccct ttcatcgccg gcaagagaat cgtgccagtg 7620
atcgagaatc acagattcac cggcagatac cgggacctgt atcctgccaa cgagctgatc 7680
gccctgctgg aggagaaggg catcgtgttc agggatggct ccaacatcct gccaaagctg 7740
ctggagaatg acgattctca cgccatcgac accatggtgg ccctgatccg cagcgtgctg 7800
cagatgcgga actccaatgc cgccacaggc gaggactata tcaacagccc cgtgcgcgat 7860
ctgaatggcg tgtgcttcga ctcccggttt cagaacccag agtggcccat ggacgccgat 7920
gccaatggcg cctaccacat cgccctgaag ggccagctgc tgctgaatca cctgaaggag 7980
agcaaggatc tgaagctgca gaacggcatc tccaatcagg actggctggc ctacatccag 8040
gagctgcgca ac 8052

Claims (10)

1. A double-stranded nucleic acid-based paper folding structure, which is formed by self-assembly of a double-stranded nucleic acid and a gene editing complex;
the gene editing complex comprises a gene editing protein and a gRNA, and the gRNA is complementarily combined with a recognition sequence on a double-stranded nucleic acid;
the gene editing complex folds the double-stranded nucleic acid into a folded paper structure.
2. The origami structure of claim 1, wherein said double stranded nucleic acid comprises a linear double stranded nucleic acid and/or a circular double stranded nucleic acid, preferably a linear plasmid and/or a circular plasmid;
preferably, the double-stranded nucleic acid contains the original spacer sequence;
preferably, the double stranded nucleic acid comprises NGG;
preferably, the double-stranded nucleic acid comprises TTTN.
3. The paper folding structure according to claim 1 or 2, wherein the gene-editing proteins comprise dCas proteins, preferably dCas9 and dCas12a, and the dCas9 and dCas12a are linked by a linker peptide to form a fusion protein.
4. The paper folding structure of any one of claims 1 to 3 wherein the gRNA includes a first gRNA and a second gRNA that are complementarily bound to adjacent recognition sequences of spaced original spacer sequences within a double-stranded nucleic acid;
preferably, the first gRNA directs dCas9 to bind to the NGG site of a double-stranded nucleic acid and the second gRNA directs dCas12a to bind to the TTTN site of a double-stranded nucleic acid.
5. The origami structure according to any one of claims 1 to 4, wherein said double stranded nucleic acid comprises a nucleic acid sequence as set forth in one of SEQ ID NO 1 to 4;
SEQ ID NO:1：
ttacgccgaagcagaatactgtgagaactcaaggtcagtagagtcatccgctgattacattcgcagttagtataggtcagtagagtcatccattcactgtatagattctcatacatggtcagtagagtcatcctgttatcactcgctgagcatagaatggtcagtagagtcatcctcactcagagtggaagaagagaataggtcagtagagtcatcctgcgcgtcgctgttagcgatgcagtggtcagtagagtcatcctgcgcacatgacgcacttgtaacgcggtcagtagagtcatccctacactcgtgcacagcgcgatcacggtcagtagagtcatccacgtaatatgagctgagctactcgtggtcagtagagtcatccagcgacagcgatcatctgaacgtatggtcagtagagtcatccagctcgtgatatattgtgcgatcatggtcagtagagtcatcctacatcgagatgactcgctgatgtaggtcagtagagtcatccggcaacagagttgcacgcacgctatggtcagtagagtcatccttatagctacgctcaagtgagctgtggtcagtagagtcatccacgtgattagagatgcgctgtatacggtcagtagagtcatccgatcgttcgacgaacagttacgcgcggtcagtagagtcatcctcgtgttcgagatctatgaattattggtcagtagagtcatccggattcgtacgtgcgagattgtcttggtcagtagagtcgtttacgctctcgcatataatcgcgagttaaacagtagagtcgtttacacgcgagcattctagtagcgcgtaaacagtagagtcgtttagcattagtgagtgacaatgctattaaacagtagagtcgtttacttcagtagtcacgatagtatcgtaaacagtagagtcgtttagtgatagttaacgctgtatgtgctaaacagtagagtcgtttagtcgagacaataagcgtgtatagtaaacagtagagtcgtttagctagttattcatcacatacacttaaacagtagagtcgtttagcacgtacatgtataactagcgttaaacagtagagtcgtttacgctataatagtgcgttgccgtctaaacagtagagtcgtttagagcgactctcgcgcagcacgcgtaaacagtagagtcgtttaattatcacgctgattcttgtaactaaacagtagagtcgtttaatactatcgattgatctcgaagctaaacagtagagtcgtttagttcagatgaatcgatattgaactaaacagtagagtcgtttacgtgatatcaagcgctgcgtgcgtaaacagtagagtcgtttagcagcatcacgatgagagctgtataaacagtagagtcgtttatgtgcatgtgtgctaatagtgagtaaacagtagagtcgtttaagagcgagtgttcagcttcatgttaaacagtagagtcgtttaacgatgctatagaactcaagcagtaaacgctt；
SEQ ID NO:2：
tatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttacgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacaccaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagaggtcgtttagtgaaccgtcagatcactagtagctttattgcggtagtttatcacagttaaattgctaacgcagtcagtgctcgactgatcacaggtaagtatcaaggttacaagacaggtttaaggaggccaatagaaactgggcttgtcgagacagagaagattcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggggtaccgaagccgctagcgctaccggtcgccaccatgcccgccatgaagatcgagtgccgcatcaccggcaccctgaacggcgtggagttcgagctggtgggcggcggagagggcacccccgagcagggccgcatgaccaacaagatgaagagcaccaaaggcgccctgaccttcagcccctacctgctgagccacgtgatgggctacggcttctaccacttcggcacctaccccagcggctacgagaaccccttcctgcacgccatcaacaacggcggctacaccaacacccgcatcgagaagtacgaggacggcggcgtgctgcacgtgagcttcagctaccgctacgaggccggccgcgtgatcggcgacttcaaggtggtgggcaccggcttccccgaggacagcgtgatcttcaccgacaagatcatccgcagcaacgccaccgtggagcacctgcaccccatgggcgataacgtgctggtgggcagcttcgcccgcaccttcagcctgcgcgacggcggctactacagcttcgtggtggacagccacatgcacttcaagagcgccatccaccccagcatcctgcagaacgggggccccatgttcgccttccgccgcgtggaggagctgcacagcaacaccgagctgggcatcgtggagtaccagcacgccttcaagacccccatcgccttcgccagatctcgagctcgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtacttaagagggggagaccaaagggcgagacgttaaggcctcacgtgacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacgggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgccgtctcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgctcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctctctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagtacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcttgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttcaatcataatattattgaagcatttatcagggttcgtctcgtcccggtctcctcccatgcatgtcaatattggccattagccatattattcattggttatatagcataaatcaatattggctattggccattgcatacgttgtatctatatcataatatgtacatttatattggctcatgtccaatatgaccgccatgttggcattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccata；
SEQ ID NO:3：
gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgggtcttcgagaagacctgtttaagagctatgctggaaacagcatagcaagtttaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagctagaaatagcaagttaaaataaggctagtccgtagcgcgtgcgccaattctgcagacaaatggctctagagttaacggatccaagcttgtcgacggtacccgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttggaccggtgaattcctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccgctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaggaccgggatgcagaaattgatgatctattaaacaataaagatgtccactaaaatggaagtttttcctgtcatactttgttaagaagggtgagaacagagtacctacattttgaatggaaggattggagctacgggggtgggggtggggtgggattagataaatgcctgctctttactgaaggctctttactattgctttatgataatgtttcatagttggatatcataatttaaacaagcaaaaccaaattaagggccagctcattcctcccactcatgatctatagatctatagatctctcgtgggatcattgtttttctcttgattcccactttgtggttctaagtactgtggtttccaaatgtgtcagtttcatagcctgaagaacgagatcagcagcctctgttccacatacacttcattctcagtattgttttgccaagttctaattccatcagaagctggtcgacctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtctcgaggctagcacgcgt；
SEQ ID NO:4：
gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgggtcttcgagaagacctgtttaagagctatgctggaaacagcatagcaagtttaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagctagaaatagcaagttaaaataaggctagtccgtagcgcgtgcgccaattctgcagacaaatggctctagagttaacggatccaagcttgtcgacggtacccgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccccaaagaagaagcggaaggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggcaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaagaattcctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccgctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaggatccgggatgcagaaattgatgatctattaaacaataaagatgtccactaaaatggaagtttttcctgtcatactttgttaagaagggtgagaacagagtacctacattttgaatggaaggattggagctacgggggtgggggtggggtgggattagataaatgcctgctctttactgaaggctctttactattgctttatgataatgtttcatagttggatatcataatttaaacaagcaaaaccaaattaagggccagctcattcctcccactcatgatctatagatctatagatctctcgtgggatcattgtttttctcttgattcccactttgtggttctaagtactgtggtttccaaatgtgtcagtttcatagcctgaagaacgagatcagcagcctctgttccacatacacttcattctcagtattgttttgccaagttctaattccatcagaagctggtcgacctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtctcgaggctagcacgcgt；
preferably, the gRNA comprises a nucleic acid sequence shown in SEQ ID NO. 5-126;
preferably, the gene-editing protein comprises the nucleic acid sequence shown in SEQ ID NO: 127;
SEQ ID NO:127：
gacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggcaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggacgccatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgacggtggcggcggtagcggtggcggtggcagcatgacacagttcgagggctttaccaacctgtatcaggtgagcaagacactgcggtttgagctgatcccacagggcaagaccctgaagcacatccaggagcagggcttcatcgaggaggacaaggcccgcaatgatcactacaaggagctgaagcccatcatcgatcggatctacaagacctatgccgaccagtgcctgcagctggtgcagctggattgggagaacctgagcgccgccatcgactcctatagaaaggagaaaaccgaggagacaaggaacgccctgatcgaggagcaggccacatatcgcaatgccatccacgactacttcatcggccggacagacaacctgaccgatgccatcaataagagacacgccgagatctacaagggcctgttcaaggccgagctgtttaatggcaaggtgctgaagcagctgggcaccgtgaccacaaccgagcacgagaacgccctgctgcggagcttcgacaagtttacaacctacttctccggcttttatgagaacaggaagaacgtgttcagcgccgaggatatcagcacagccatcccacaccgcatcgtgcaggacaacttccccaagtttaaggagaattgtcacatcttcacacgcctgatcaccgccgtgcccagcctgcgggagcactttgagaacgtgaagaaggccatcggcatcttcgtgagcacctccatcgaggaggtgttttccttccctttttataaccagctgctgacacagacccagatcgacctgtataaccagctgctgggaggaatctctcgggaggcaggcaccgagaagatcaagggcctgaacgaggtgctgaatctggccatccagaagaatgatgagacagcccacatcatcgcctccctgccacacagattcatccccctgtttaagcagatcctgtccgataggaacaccctgtctttcatcctggaggagtttaagagcgacgaggaagtgatccagtccttctgcaagtacaagacactgctgagaaacgagaacgtgctggagacagccgaggccctgtttaacgagctgaacagcatcgacctgacacacatcttcatcagccacaagaagctggagacaatcagcagcgccctgtgcgaccactgggatacactgaggaatgccctgtatgagcggagaatctccgagctgacaggcaagatcaccaagtctgccaaggagaaggtgcagcgcagcctgaagcacgaggatatcaacctgcaggagatcatctctgccgcaggcaaggagctgagcgaggccttcaagcagaaaaccagcgagatcctgtcccacgcacacgccgccctggatcagccactgcctacaaccctgaagaagcaggaggagaaggagatcctgaagtctcagctggacagcctgctgggcctgtaccacctgctggactggtttgccgtggatgagtccaacgaggtggaccccgagttctctgcccggctgaccggcatcaagctggagatggagccttctctgagcttctacaacaaggccagaaattatgccaccaagaagccctactccgtggagaagttcaagctgaactttcagatgcctacactggcctctggctgggacgtgaataaggagaagaacaatggcgccatcctgtttgtgaagaacggcctgtactatctgggcatcatgccaaagcagaagggcaggtataaggccctgagcttcgagcccacagagaaaaccagcgagggctttgataagatgtactatgactacttccctgatgccgccaagatgatcccaaagtgcagcacccagctgaaggccgtgacagcccactttcagacccacacaacccccatcctgctgtccaacaatttcatcgagcctctggagatcacaaaggagatctacgacctgaacaatcctgagaaggagccaaagaagtttcagacagcctacgccaagaaaaccggcgaccagaagggctacagagaggccctgtgcaagtggatcgacttcacaagggattttctgtccaagtataccaagacaacctctatcgatctgtctagcctgcggccatcctctcagtataaggacctgggcgagtactatgccgagctgaatcccctgctgtaccacatcagcttccagagaatcgccgagaaggagatcatggatgccgtggagacaggcaagctgtacctgttccagatctataacaaggactttgccaagggccaccacggcaagcctaatctgcacacactgtattggaccggcctgttttctccagagaacctggccaagacaagcatcaagctgaatggccaggccgagctgttctaccgccctaagtccaggatgaagaggatggcacaccggctgggagagaagatgctgaacaagaagctgaaggatcagaaaaccccaatccccgacaccctgtaccaggagctgtacgactatgtgaatcacagactgtcccacgacctgtctgatgaggccagggccctgctgcccaacgtgatcaccaaggaggtgtctcacgagatcatcaaggataggcgctttaccagcgacaagttctttttccacgtgcctatcacactgaactatcaggccgccaattccccatctaagttcaaccagagggtgaatgcctacctgaaggagcaccccgagacacctatcatcggcatcgatcggggcgagagaaacctgatctatatcacagtgatcgactccaccggcaagatcctggagcagcggagcctgaacaccatccagcagtttgattaccagaagaagctggacaacagggagaaggagagggtggcagcaaggcaggcctggtctgtggtgggcacaatcaaggatctgaagcagggctatctgagccaggtcatccacgagatcgtggacctgatgatccactaccaggccgtggtggtgctggagaacctgaatttcggctttaagagcaagaggaccggcatcgccgagaaggccgtgtaccagcagttcgagaagatgctgatcgataagctgaattgcctggtgctgaaggactatccagcagagaaagtgggaggcgtgctgaacccataccagctgacagaccagttcacctcctttgccaagatgggcacccagtctggcttcctgttttacgtgcctgccccatatacatctaagatcgatcccctgaccggcttcgtggaccccttcgtgtggaaaaccatcaagaatcacgagagccgcaagcacttcctggagggcttcgactttctgcactacgacgtgaaaaccggcgacttcatcctgcactttaagatgaacagaaatctgtccttccagaggggcctgcccggctttatgcctgcatgggatatcgtgttcgagaagaacgagacacagtttgacgccaagggcacccctttcatcgccggcaagagaatcgtgccagtgatcgagaatcacagattcaccggcagataccgggacctgtatcctgccaacgagctgatcgccctgctggaggagaagggcatcgtgttcagggatggctccaacatcctgccaaagctgctggagaatgacgattctcacgccatcgacaccatggtggccctgatccgcagcgtgctgcagatgcggaactccaatgccgccacaggcgaggactatatcaacagccccgtgcgcgatctgaatggcgtgtgcttcgactcccggtttcagaacccagagtggcccatggacgccgatgccaatggcgcctaccacatcgccctgaagggccagctgctgctgaatcacctgaaggagagcaaggatctgaagctgcagaacggcatctccaatcaggactggctggcctacatccaggagctgcgcaac。
6. a method of making the origami structure of any of claims 1-5, comprising:
and constructing a gene editing compound, and mixing and incubating the gene editing compound with double-stranded nucleic acid to obtain the paper folding structure.
7. The method of claim 6, wherein the gene-editing complex is prepared by:
and mixing and incubating the gene editing protein, the first gRNA and the second gRNA to form a gene editing complex.
8. The method of claim 7, wherein the molar ratio of the gene-editing protein to the first gRNA to the second gRNA is 1 (1-1.5): 1-1.5);
preferably, the incubation temperature is 30-37 ℃;
preferably, the incubation time is 20-40 min.
9. The method according to any one of claims 6 to 8, wherein the molar ratio of the gene-editing complex to the double-stranded nucleic acid is 1 (1 to 1.5);
preferably, the incubation temperature is 30-37 ℃;
preferably, the incubation time is 20-40 min.
10. Use of the origami structure of any of claims 1-5 to increase the stability of double stranded nucleic acids.
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