CN106916852A - 一种碱基编辑系统及其构建和应用方法 - Google Patents
一种碱基编辑系统及其构建和应用方法 Download PDFInfo
- Publication number
- CN106916852A CN106916852A CN201710239348.XA CN201710239348A CN106916852A CN 106916852 A CN106916852 A CN 106916852A CN 201710239348 A CN201710239348 A CN 201710239348A CN 106916852 A CN106916852 A CN 106916852A
- Authority
- CN
- China
- Prior art keywords
- ugi
- base editing
- dna
- base
- expression vectors
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 45
- 239000013604 expression vector Substances 0.000 claims abstract description 51
- 108091033409 CRISPR Proteins 0.000 claims abstract description 43
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 42
- 239000013598 vector Substances 0.000 claims abstract description 13
- 108020004414 DNA Proteins 0.000 claims description 73
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 22
- 239000003153 chemical reaction reagent Substances 0.000 claims description 14
- 238000001712 DNA sequencing Methods 0.000 claims description 13
- 238000013518 transcription Methods 0.000 claims description 13
- 230000035897 transcription Effects 0.000 claims description 13
- 230000004907 flux Effects 0.000 claims description 12
- 238000012408 PCR amplification Methods 0.000 claims description 10
- 102100037964 E3 ubiquitin-protein ligase RING2 Human genes 0.000 claims description 8
- 101001095815 Homo sapiens E3 ubiquitin-protein ligase RING2 Proteins 0.000 claims description 8
- 230000014509 gene expression Effects 0.000 claims description 6
- 238000010276 construction Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 2
- 230000002708 enhancing effect Effects 0.000 claims 2
- 101150025342 UGI gene Proteins 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 34
- 238000003780 insertion Methods 0.000 abstract description 17
- 230000037431 insertion Effects 0.000 abstract description 17
- 239000002585 base Substances 0.000 description 116
- 239000013613 expression plasmid Substances 0.000 description 27
- 210000004027 cell Anatomy 0.000 description 15
- 239000013612 plasmid Substances 0.000 description 15
- 238000001890 transfection Methods 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 8
- 101150063416 add gene Proteins 0.000 description 7
- 210000003527 eukaryotic cell Anatomy 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000010362 genome editing Methods 0.000 description 6
- 102000012410 DNA Ligases Human genes 0.000 description 5
- 230000006798 recombination Effects 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 239000012096 transfection reagent Substances 0.000 description 5
- 108010061982 DNA Ligases Proteins 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 230000004087 circulation Effects 0.000 description 4
- 238000012937 correction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000009977 dual effect Effects 0.000 description 4
- 238000013326 plasmid cotransfection Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000010453 CRISPR/Cas method Methods 0.000 description 3
- 108020005004 Guide RNA Proteins 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 238000011022 operating instruction Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 241000206602 Eukaryota Species 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 230000004186 co-expression Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 208000016361 genetic disease Diseases 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108010040467 CRISPR-Associated Proteins Proteins 0.000 description 1
- 102000000311 Cytosine Deaminase Human genes 0.000 description 1
- 108010080611 Cytosine Deaminase Proteins 0.000 description 1
- 108010076804 DNA Restriction Enzymes Proteins 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 230000008265 DNA repair mechanism Effects 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 101000595467 Homo sapiens T-complex protein 1 subunit gamma Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102100036049 T-complex protein 1 subunit gamma Human genes 0.000 description 1
- 102100037111 Uracil-DNA glycosylase Human genes 0.000 description 1
- 101710172430 Uracil-DNA glycosylase inhibitor Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000003878 glomerular mesangial cell Anatomy 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000006780 non-homologous end joining Effects 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种碱基编辑系统及其构建和应用方法。所述的碱基编辑系统，其特征在于，包括UGI表达载体和BE3表达载体，或包括UGI和BE3共表达载体。本发明在国际上首次通过增加CRISPR碱基编辑器产生的C‑T编辑率并降低CRISPR系统本身引起的碱基插入或缺失率，从而增强了CRISPR碱基编辑器的功效，为CRISPR碱基编辑器在各物种的基因组中实施更为准确和安全的碱基编辑提供了新的方法和思路。
Description
技术领域
本发明涉及基因组编辑领域，具体涉及一种增强的CRISPR碱基编辑系统实现高效率和高精度的碱基水平基因组编辑方法及其应用。
背景技术
基因组编辑技术指利用可设计的核酸酶(分子剪刀)通过碱基插入、缺失或置换等方式，对生物体基因组DNA特定片段进行改造从而达到对目标基因进行编辑的一种基因工程技术。利用基因组编辑技术对细胞进行遗传学操纵，可广泛的应用于生命科学基础研究、生物技术开发、农业技术开发以及医药研发领域。例如：直接在体内校正引起遗传疾病的基因突变，将能够从根本上治疗遗传疾病；对农作物进行精确的基因工程改造，使其产量提高或能够抵抗环境污染或病原体的感染；对微生物基因组进行精准改造，从而促进可再生生物能源的开发等。
CRISPR/Cas(Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)基因组编辑系统自问世以来，就有着其它基因组编辑技术无可比拟的优势，被认为能够在活细胞中广泛使用，并且是迄今为止最有效、最便捷的基因组编辑系统。Cas9核酸酶利用引导RNA(guide RNA，gRNA)可在多种细胞的基因组特定靶点定位，对其进行切割从而产生DNA双链断裂(double strand breaks，DSBs)，然后利用细胞内源的DNA修复机制来实现编辑。根据不同DNA修复通路的激活，基因组编辑将会导致基因的失活或者突变的校正。通常来说，有两种主要的修复机制会被DSB所激活，一种是非同源末端连接(non-homologous end joining，NHEJ)，另一种是同源性介导修复(homology-directed repair，HDR)。做为DNA双链断裂最主要的修复通路，NHEJ在修复过程中能在DSB附近的基因组位点引入随机性碱基插入或缺失，从而导致基因的失活。与NHEJ相反，当HDR被激活时能够以外源性供体DNA作为模版，利用同源重组机制将外源性供体DNA的序列替换靶点基因组DNA的序列，从而完成基因突变的校正。但实际上，由于同源重组机制本身的局限性，HDR介导的基因校正效率一直很低(通常小于5％)。因此，极大限制了CRISPR/Cas基因组编辑工具从科研向应用的转化，尤其是在精准基因治疗方面的应用，这也是基因编辑领域的一大难题。
为了提高基因突变的校正效率，碱基编辑器(base editor，BE)在近期内应运而生。这种碱基编辑器将CRISPR/Cas系统和APOBEC胞嘧啶脱氨酶家族成员整合在一起，可执行将胞嘧啶(cytosine，C)编辑为胸腺嘧啶(thymine，T)的功能。为了最大程度地提升C-T碱基编辑的效率，研究人员尝试了将完全丧失酶活性的dCas9或者具有切刻酶活性的nCas9与APOBEC整合，从而产生了碱基编辑器2(BE2)和碱基编辑器3(BE3)。相对于含有dCas9的BE2，含有nCas9的BE3虽然在靶点序列有着更高的C-T编辑效率，但BE3在靶点序列由CRISPR系统本身所引起的碱基插入或缺失(insertion/deletion，indel)率却更高，反而降低了碱基编辑的功效(C-T碱基编辑率与碱基插入或缺失率的比值，C-T editing rate/indel rate)及精确度。
因此，创造具有更高编辑功效的增强型碱基编辑系统，有助于实现高精度的碱基编辑，能极大地扩充我们对于碱基编辑器的应用，特别是在医疗领域进行精准基因治疗方面。
发明内容
本发明的目的在于克服现有技术碱基编辑功效低的缺陷，提供一种碱基编辑系统及方法，其利用共表达BE3与尿嘧啶糖苷酶抑制剂(uracil DNA glycosylase inhibitor，UGI)的方法提高碱基编辑器的功效，实现高精度的碱基编辑，从而可以更为有效的在各物种基因组中进行碱基编辑。
为了达到上述目的，本发明提供了以下技术方案：
一种碱基编辑系统，其特征在于，包括UGI表达载体或其转录产物和BE3表达载体或其转录产物，或包括UGI和BE3共表达载体或其转录产物。
优选地，所述的UGI和BE3共表达载体为增强型CRISPR碱基编辑器eBEa表达载体，其序列包括SEQ ID NO：22。
优选地，所述的UGI和BE3共表达载体为增强型CRISPR碱基编辑器eBEb表达载体，其序列包括SEQ ID NO：23。
优选地，所述的UGI表达载体的序列包括SEQ ID NO：20。
优选地，所述的BE3表达载体的序列包括SEQ ID NO：21。
优选地，所述的碱基编辑系统还包括针对某物种的基因组靶点的sgRNA表达载体。
优选地，所述的碱基编辑系统还包括基因转导试剂。
优选地，所述的碱基编辑系统还包括真核细胞。
优选地，所述的UGI表达载体、BE3表达载体以及UGI和BE3共表达载体包括但不限于质粒载体。
优选地，所述的转录产物包括但不限于体外转录出的RNA。
本发明还提供了上述的碱基编辑系统的构建方法，其特征在于，包括扩增UGI基因，并连入真核表达载体中，得到UGI表达载体；或利用PCR方法扩增T2A-UGI编码序列，并同框连入BE3编码区的下游，得到增强型CRISPR碱基编辑器eBEa表达载体；或人工合成3×T2A-UGI编码序列，PCR扩增后同框连入BE3编码区的下游，得到到增强型CRISPR碱基编辑器eBEb表达载体。
本发明还提供了上述的碱基编辑系统的应用方法，其特征在于，包括构建针对某物种的基因组DNA靶点的sgRNA；在真核细胞中共引入sgRNA，BE3表达载体或其转录产物以及UGI表达载体或其转录产物，或者，在真核细胞中共引入sgRNA以及UGI和BE3共表达载体或其转录产物，使其在靶点基因组DNA发生C-T碱基编辑。
优选地，所述的碱基编辑系统的应用方法还包括：对处理后的真核细胞基因组DNA进行抽提，然后利用针对靶点的特异性引物进行PCR扩增，利用基因组DNA的PCR产物构建高通量二代DNA测序文库，并进行高通量二代DNA测序。
优选地，所述的某物种的基因组DNA靶点包括但不限于人的FANCF和RNF2靶点中的至少一个。
优选地，所述的sgRNA的序列为SEQ ID NO：24或SEQ ID NO：25。
本发明提供了一种共表达BE3与UGI的方法，首先通过构建UGI单独表达载体载体，并与BE3表达载体和sgRNA表达载体共转染真核生物细胞，使其在靶点基因组DNA发生C-T碱基编辑，从而提高了BE3的功效。
为进一步优化实验，本发明在BE3编码框的下游利用T2A自剪切肽段同框连接1×或者3×UGI基因，从而得到增强型CRISPR碱基编辑器eBEa和eBEb表达载体，并将其与sgRNA表达载体共转染真核细胞，从而实现了高功效高精度的碱基编辑。本发明以人基因组DNA靶点FANCF(实施例1)和RNF2(实施例2)位点为例，实施了高功效高精度的C-T碱基编辑。
与现有技术相比，本发明的有益效果是：
本发明在国际上首次通过增加CRISPR碱基编辑器产生的C-T编辑率并降低CRISPR系统本身引起的碱基插入或缺失率，从而增强了CRISPR碱基编辑器的功效，为CRISPR碱基编辑器在各物种的基因组中实施更为准确和安全的碱基编辑提供了新的方法和思路。
本发明所述的单/双载体共表达CRISPR碱基编辑器与UGI的方法，可以在各种真核生物的基因组中实施高功效高精度的C-T碱基编辑。
本发明所述的单/双载体共表达CRISPR碱基编辑器和UGI的方法，可以提高多种CRISPR碱基编辑器的C-T碱基编辑率，并降低CRISPR系统本身引起的碱基插入或缺失率，从而增强多种CRISPR碱基编辑器的功效。
附图说明
图1A：实施例1双载体共表达BE3与UGI的方法示意图
图1B：实施例1双载体共表达BE3与UGI的方法提高了碱基编辑器的功效。C6、C7、C8、C11代表其靶点序列中的第6、7、8、11位的胞嘧啶。
图1C：实施例1双载体共表达BE3与UGI的方法提高了碱基编辑器的C-T编辑率。C6、C7、C8、C11代表其靶点序列中的第6、7、8、11位的胞嘧啶。
图1D：实施例1双载体共表达BE3与UGI的方法抑制了CRISPR系统本身引起的碱基插入或缺失率。
图1E：实施例1单载体共表达BE3与UGI的方法示意图
图1F：实施例1单载体共表达BE3与UGI的方法提高了碱基编辑器的功效。C6、C7、C8、C11代表其靶点序列中的第6、7、8、11位的胞嘧啶。
图1G：实施例1单载体共表达BE3与UGI的方法提高了碱基编辑器的C-T编辑率。C6、C7、C8、C11代表其靶点序列中的第6、7、8、11位的胞嘧啶。
图1H：实施例1单载体共表达BE3与UGI的方法抑制了CRISPR系统本身引起的碱基插入或缺失率。
图2A：实施例2双载体共表达BE3与UGI的方法示意图
图2B：实施例2双载体共表达BE3与UGI的方法提高了碱基编辑器的功效。C3、C6代表其靶点序列中的第3、6位的胞嘧啶。
图2C：实施例2双载体共表达BE3与UGI的方法提高了碱基编辑器的C-T编辑率。C3、C6代表其靶点序列中的第3、6位的胞嘧啶。
图2D：实施例2双载体共表达BE3与UGI的方法抑制了CRISPR系统本身引起的碱基插入或缺失率。
图2E：实施例2单载体共表达BE3与UGI的方法示意图
图2F：实施例2单载体共表达BE3与UGI的方法提高了碱基编辑器的功效。C3、C6代表其靶点序列中的第3、6位的胞嘧啶。
图2G：实施例2单载体共表达BE3与UGI的方法提高了碱基编辑器的C-T编辑率。C3、C6代表其靶点序列中的第3、6位的胞嘧啶。
图2H：实施例2单载体共表达BE3与UGI的方法抑制了CRISPR系统本身引起的碱基插入或缺失率。
具体实施方式
本发明的共表达BE3与UGI的方法，首先通过构建UGI单独表达载体载体，并与BE3表达载体和sgRNA表达载体共转染真核生物细胞，从而提高了BE3的功效。为进一步优化实验，本发明在BE3编码框的下游利用T2A自剪切肽段同框连接1×或者3×UGI基因，从而得到增强型CRISPR碱基编辑器eBEa和eBEb表达载体，并将其与sgRNA表达载体共转染真核细胞，从而实现了高功效高精度的碱基编辑。本发明以人基因组DNA靶点FANCF(实施例1)和RNF2(实施例2)位点为例，实施了高功效高精度的C-T碱基编辑。
具体的，实验方法包括下列步骤：
1)构建表达载体。
将UGI基因从BE3表达载体中利用PCR扩增出来，并连入真核表达载体pcDNA3中，得到UGI单独表达载体载体。
利用PCR方法扩增T2A-UGI编码序列，并同框连入BE3编码区的下游，得到到增强型CRISPR碱基编辑器eBEa表达载体。
人工合成3×T2A-UGI编码序列，PCR扩增后同框连入BE3编码区的下游，得到到增强型CRISPR碱基编辑器eBEb表达载体。
构建针对人基因组DNA靶点FANCF和RNF2的sgRNA。
2)转染真核细胞。
在真核细胞中共转染sgRNA，BE3表达载体以及低、中、高不同浓度的UGI表达载体。
在真核细胞中共转染sgRNA和eBEa、eBEb表达载体。
3)基因组DNA抽提扩增。
抽提转染后的真核细胞基因组DNA，然后利用针对靶点FANCF和RNF2的特异性引物进行PCR扩增。
4)基因组DNAPCR产物的高通量二代DNA测序。
利用基因组DNA的PCR产物构建高通量二代DNA测序文库，并进行高通量二代DNA测序。
本发明所述的单/双载体共表达CRISPR碱基编辑器与UGI的方法，可以在各种真核生物的基因组中实施高功效高精度的C-T碱基编辑。
本发明所述的单/双载体共表达CRISPR碱基编辑器和UGI的方法，可以提高多种CRISPR碱基编辑器的C-T碱基编辑率，并降低CRISPR系统本身引起的碱基插入或缺失率，从而增强多种CRISPR碱基编辑器的功效。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、细胞生物学、重组DNA技术及相关领域的常规技术，这些技术在现有文献中已有完善说明。
实施例1
一种碱基编辑系统，包括UGI表达质粒(序列为SEQ ID NO：20)和BE3表达质粒(序列为SEQ ID NO：21)，或包括UGI和BE3共表达质粒。所述的UGI和BE3共表达质粒为增强型CRISPR碱基编辑器eBEa表达质粒(序列为SEQ ID NO：22)或增强型CRISPR碱基编辑器eBEb表达质粒(序列为SEQ ID NO：23)。
人基因组FANCF位点利用上述的碱基编辑系统实施高功效高精度的碱基编辑：
一.实验材料
1)试剂
引物按常规方法合成自上海博尚生物有限公司；
限制性内切酶、DNA连接酶、高保真DNA聚合酶购自NEB公司；
质粒重组试剂盒Clone购自Vazyme公司；
pCMV-BE3购自addgene网站，序列为SEQ ID NO：21；
DNA胶回收试剂盒购自Coming公司；
转染试剂2000，购自Thermo Fisher公司；
QuickExtractTM基因组DNA抽提试剂、高通量DNA二代测序文库构建试剂盒TmSeqChIPLibary Preparation Kit购自Illumina公司。
2)细胞株
人胎肾细胞HEK293FT(ThermoFisher Scientific，R70007)在加入了1ml含有1％双抗的DMEM(Gibco，10566)+10％FBS(Gbico，16000-044)培养基的6孔板中贴壁培养至单孔细胞个数为八十万。
二.实验方法
1、碱基编辑系统的构建
1.1 UGI单独表达质粒的构建
使用以下引物1和含有核定位序列(NLS)的引物2将UGI基因从pCMV-BE3质粒(addgene，#73021)中PCR扩增出来，之后用质粒重组试剂盒Clone(Vazyme，C112-02)按试剂盒操作说明将下游连有NLS的UGI基因重组至真核表达载体pcDNA3.1(+)(ThermoFisher Scientific，V79020)中，得到UGI单独表达质粒pUGI-NLS(序列为SEQ IDNO：20)。
1.2 eBEa、eBEb表达质粒的构建
利用以下引物3和引物4以pCMV-BE3质粒(addgene，#73021)为模版进行PCR扩增，将PCR产物用质粒重组试剂盒Clone(Vazyme，C112-02)按试剂盒操作说明重组至pCMV-BE3质粒(addgene，#73021)中得到含有Apa I(NEB，R0114S)、Pme I(NEB，R0560L)酶切位点的质粒pCMV-BE3AP。利用以下引物5和引物6以化学合成的DNA序列(序列为SEQ ID NO：19)为模版进行PCR扩增，将PCR产物经限制性内切酶Apa I(NEB，R0114S)、Pme I(NEB，R0560L)消化后利用T4 DNA连接酶(NEB，M0202L)按试剂说明连入同样经限制性内切酶ApaI(NEB，R0114S)、Pme I(NEB，R0560L)消化后的pCMV-BE3AP(25℃，2hr)，得到eBEb表达质粒pCMV-eBEb(序列为SEQ ID NO：23)。
利用以下引物3，引物7，引物8和引物9以pCMV-BE3为模版进行PCR扩增，将两段PCR产物用质粒重组试剂盒Clone(Vazyme，C112-02)按试剂盒操作说明重组至pCMV-BE3质粒(addgene，#73021)中得到pCMV-BE3T。
利用以下引物5和引物10以pCMV-BE3T为模版进行PCR扩增，将PCR产物经限制性内切酶Apa I(NEB，R0114S)、Pme I(NEB，R0560L)消化后利用T4 DNA连接酶(NEB，M0202L)按试剂说明连入同样经限制性内切酶Apa I(NEB，R0114S)、Pme I(NEB，R0560L)消化后的pCMV-BE3AP(25℃，2hr)，得到eBEa表达质粒pCMV-eBEa(序列为SEQ ID NO：22)。
2、碱基编辑系统的应用
2.1 sgRNA表达质粒的构建
将以下引物11和引物12退火，将退火产物利用T4 DNA连接酶(NEB，M0202L)按照试剂说明连入经限制性内切酶BsaI(NEB，R3535L)消化后的sgRNA表达载体pGL3-U6-sgRNA-PGK-puromycin(addgene，#51133)后，得到靶向人基因组FANCF位点的sgRNA表达质粒psgFANCF(序列为SEQ ID NO：24)。
表1引物
步骤1.2、1.2和2.1中质粒构建实验中扩增各种片段使用PCR体系如下：
Phusion(NEB，M0535L)1μl；5×buffer(NEB，B0518S)4μl；Forward Primer(5μM)1μl；Reverse Primer(5μM)1μl；Template10ng；RNase-free water补至50μl。PCR程序如下：95℃，2min；95℃，15sec，55℃，30sec，72℃，1min，30循环；72℃，3min。
2.2真核细胞转染
2.2.1双载体共表达BE3和UGI
将psgFANCF、pCMV-BE3和得到的序列正确的pUGI-NLS质粒按照1.6μg/2.5μg/0ng(BE3_no UGI)，1.6μg/2.5μg/50ng(BE3_low UGI)，1.6μg/2.5μg/100ng(BE3_medium UGI)，1.6μg/2.5μg/200ng(BE3_highUGI)，0μg/0μg/0ng(no BE3)的比例混合入250μlOpti-MEM中，再混入含有2000(Thermo Fisher，15338100)转染试剂的250μlOpti-MEM(Thermo Fisher，51985-042)，室温放置30分钟后加入含有1ml 1％双抗的DMEM(Gibco，10566)+10％FBS(Gbico，16000-044)培养基和八十万HEK293FT细胞的6孔板中进行转染。转染24h后换含有1ug/ml puromycine(Invivogen，ant-pr-1)抗生素的新鲜培养基，继续培养48小时。
2.2.2单载体共表达BE3和UGI
将psgFANCF和pCMV-BE3(或得到的序列正确的pCMV-eBEa或pCMV-eBEb)质粒按照1.6μg/2.5μg的比例混合入250μlOpti-MEM中，再混入含有2000(ThermoFisher，15338100)转染试剂的250μlOpti-MEM(Thermo Fisher，51985-042)，室温放置30分钟后加入含有1ml 1％双抗的DMEM(Gibco，10566)+10％FBS(Gbico，16000-044)培养基和八十万HEK293FT细胞的6孔板中进行转染。转染24h后换含有1ug/ml puromycine(Invivogen，ant-pr-1)抗生素的新鲜培养基，继续培养48小时。
2.3基因组DNA抽提和PCR扩增
利用QuickExtractTM(Epicenter/Illumina，QE09050)试剂按照试剂操作说明抽提转染后HEK293FT细胞的基因组DNA，将抽提得到的基因组DNA利用以下引物13和引物14进行PCR扩增，并利用DNA胶回收试剂(AP-GX-500G)按照试剂操作说明对PCR产物进行切胶回收，用于后续的高通量二代DNA测序文库的构建。
表2引物
| 编号 | 引物名 | 序列 |
| 13 | hFANCF_GC1F | cacagtatgtctctggcgtt |
| 14 | hFANCF_GC1R | tctcttggagtgtctcctca |
扩增基因组DNA使用PCR体系如下：
Phusion(NEB，M0535L)1μl；5×buffer(NEB，B0518S)4μl；Forward Primer(5μM)1μl；Reverse Primer(5μM)1μl；Template10ng；RNase-free water补至50μl。PCR程序如下：95℃，2min；95℃，15sec，55℃，15sec，72℃，30sec，30循环；72℃，3min。
2.4高通量二代DNA测序
对PCR产物进行DNA-seq的高通量测序文库制备。具体来说，首先分别使用QubitFluorometer和AglientBioanalyzer 2100对PCR产物进行浓度质检和纯度鉴定；质检合格的样品，再使用Covaris S220超声波DNA破碎仪将PCR产物进行片段化；然后利用Illumina官方试剂盒TruSeqChIP Sample Preparation Kit，对不同的PCR产物加上不同生物素标记barcode，进行PCR扩增富集，完成测序文库的制备，并对已经构建完成的文库进行浓度和片段大小质检；最后使用IlluminaHiseq 2500测序仪进行高通量测序。
2.5实验结果
通过将sgFANCF表达质粒、BE3表达质粒和UGI表达质粒共转染真核生物细胞(图1A)，使其在靶点基因组DNA发生C-T碱基编辑，并在PCR扩增靶点基因组DNA之后，利用高通量二代DNA测序检测sgFANCF靶点基因组DNA发生的C-T碱基编辑率和碱基插入或缺失率，最终计算所述系统的碱基编辑功效。和未表达UGI的对照组相比，共表达UGI能够在sgFANCF靶点基因组DNA的4个C位点提高碱基编辑器的功效，并且其功效随着UGI转染量的增加而提高(图1B)。同时，sgFANCF靶点基因组DNA的4个C位点的C-T碱基编辑率也得到提高(图1C)，而CRISPR系统本身所引起的碱基插入或缺失率则得到抑制(图1D)。
进一步的，本发明利用T2A自剪切肽段在BE3编码区下游同框接入1×或者3×UGI基因，得到增强型碱基编辑器a(enhanced base editor，eBEa)和增强型碱基编辑器b(eBEb)。在将sgFANCF表达质粒和BE3、eBEa或eBEb表达质粒共转染真核细胞之后(图1E)，按照上述方法检测sgFANCF靶点基因组DNA发生的C-T碱基编辑率和碱基插入或缺失率，并计算增强型碱基编辑器的功效。和BE3相比，eBEa和eBEb的功效均得到提高，并且同框接入3×UGI基因的eBEb功效为最高(图1F)。同时，eBEa和eBEb在sgFANCF靶点基因组DNA的4个C位点所诱导的C-T碱基编辑率也比BE3更高(图1G)，而CRISPR系统本身所引起的碱基插入或缺失率则较BE3更低(图1H)。
实施例2
人基因组RNF2位点利用实施例1中的碱基编辑系统实施高功效高精度的碱基编辑：
1.实验材料
1)试剂
同实施例1。
2)细胞株和载体
同实施例1。
2.实验方法
1、碱基编辑系统的构建
1.1 UGI单独表达质粒的构建
同实施例1。
1.2 eBEa、eBEb表达质粒的构建
同实施例1。
2、碱基编辑系统的应用
2.1 sgRNA表达质粒的构建
将以下引物15和引物16退火，将退火产物利用T4 DNA连接酶(NEB，M0202L)按照试剂说明连入经限制性内切酶BsaI(NEB，R3535L)消化后的sgRNA表达载体pGL3-U6-sgRNA-PGK-puromycin(addgene，#51133)后，得到靶向人基因组RNF2位点的sgRNA表达质粒psgRNF2(序列为SEQ ID NO：25)。
表3引物
| 编号 | 引物名 | 序列 |
| 15 | fwd_RNF2 | accggtcatcttagtcattacctg |
| 16 | rev_RNF2 | aaaccaggtaatgactaagatgac |
质粒构建实验中扩增各种片段使用PCR体系如下：
Phusion(NEB，M0535L)1μl；5×buffer(NEB，B0518S)4μl；Forward Primer(5μM)1μl；Reverse Primer(5μM)1μl；Template10ng；RNase-free water补至50μl。PCR程序如下：95℃，2min；95℃，15sec，55℃，30sec，72℃，1min，30循环；72℃，3min。
2.2真核细胞转染
2.2.1双载体共表达BE3和UGI
将psgRNF2、pCMV-BE3和得到的序列正确的pUGI-NLS质粒按照1.6μg/2.5μg/0ng(BE3_no UGI)，1.6μg/2.5μg/50ng(BE3_low UGI)，1.6μg/2.5μg/100ng(BE3_medium UGI)，1.6μg/2.5μg/200ng(BE3_high UGI)，0μg/0μg/0ng(no BE3)的比例混合入250μlOpti-MEM(Thermo Fisher，51985-042)中，再混入含有2000(Thermo Fisher，15338100)转染试剂的250μlOpti-MEM，室温放置30分钟后加入含有1ml DMEM DMEM(Gibco，10566)+10％FBS(Gbico，16000-044)培养基和八十万HEK293FT细胞的6孔板中进行转染。转染24h后换含有1ug/ml puromycine(Invivogen，ant-pr-1)抗生素的新鲜培养基，继续培养48小时。
2.2.1双载体共表达BE3和UGI
将psgRNF2和pCMV-BE3(或得到的序列正确的pCMV-eBEa或pCMV-eBEb)质粒按照1.6μg/2.5μg的比例混合入250μlOpti-MEM(Thermo Fisher，51985-042)中，再混入含有2000(Thermo Fisher，15338100)转染试剂的250μlOpti-MEM，室温放置30分钟后加入含有1ml 1％双抗的DMEM(Gibco，10566)+10％FBS(Gbico，16000-044)培养基和八十万HEK293FT细胞的6孔板中进行转染。转染24h后换含有1ug/ml puromycine(Invivogen，ant-pr-1)抗生素的新鲜培养基，继续培养48小时。
2.3基因组DNA抽提和PCR扩增
利用QuickExtractTM试剂按试剂操作说明抽提转染后HEK293FT细胞的基因组DNA，将抽提得到的基因组DNA利用以下引物17和引物18进行PCR扩增，并利用DNA胶回收试剂按试剂操作说明对PCR产物进行切胶回收，用于后续的高通量二代DNA测序文库的构建。
表4引物
| 编号 | 引物名 | 序列 |
| 17 | hRNF2_G2F | aggaggacttgcccaacttt |
| 18 | hRNF2_G2R | ctgtgtcagaacatgctggaa |
扩增基因组DNA使用PCR体系如下：
Phusion(NEB，M0535L)1μl；5×buffer(NEB，B0518S)4μl；Forward Primer(5μM)1μl；Reverse Primer(5μM)1μl；Template10ng；RNase-free water补至50μl。PCR程序如下：95℃，2min；95℃，15sec，55℃，15sec，72℃，30sec，30循环；72℃，3min。
2.4高通量二代DNA测序
同实施例1。
2.5实验结果
通过将sgRNF2表达质粒、BE3表达质粒和UGI表达质粒共转染真核生物细胞(图2A)，使其在靶点基因组DNA发生C-T碱基编辑，并在PCR扩增靶点基因组DNA之后，利用高通量二代DNA测序检测sgRNF2靶点基因组DNA发生的C-T碱基编辑率和碱基插入或缺失率，最终计算所述系统的碱基编辑功效。和未表达UGI的对照组相比，共表达UGI能够在sgRNF2靶点基因组DNA的2个C位点提高碱基编辑器的功效，并且其功效随着UGI转染量的增加而提高(图2B)。同时，sgRNF2靶点基因组DNA的2个C位点的C-T碱基编辑率也得到提高(图2C)，而CRISPR系统本身所引起的碱基插入或缺失率则得到抑制(图2D)。
进一步的，在将sgRNF2表达质粒和BE3、eBEa或eBEb表达质粒共转染真核细胞之后(图2E)，按照上述方法检测sgRNF2靶点基因组DNA发生的C-T碱基编辑率和碱基插入或缺失率，并计算增强型碱基编辑器的功效。和BE3相比，eBEa和eBEb的功效均得到提高，并且同框接入3×UGI基因的eBEb功效为最高(图2F)。同时，eBEa和eBEb在sgRNF2靶点基因组DNA的2个C位点所诱导的C-T碱基编辑率也比BE3更高(图2G)，而CRISPR系统本身所引起的碱基插入或缺失率则较BE3更低(图2H)。
<110>上海科技大学
<120>一种碱基编辑系统及其构建和应用方法
<130> XXX
<160> 25
<170> PatentIn version 3.5
<210> 1
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 1
gtgctggata tctgcagaat tcatgactaa tctgtcagat at 42
<210> 2
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 2
ttggtaccga gctcggatcc ctagactttc ctcttcttct tggggagcat cttaatctt 59
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 3
aaatggccga aaacggatgt 20
<210> 4
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 4
gaggctgatc agcgggttta aactcaatgg tgagggcccg actttcctct tcttcttgg 59
<210> 5
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 5
aaagtcgggc ccgagggcag aggaagtctg ct 32
<210> 6
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 6
cagcgggttt aaactcacac ctttcttttt tttttagg 38
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 7
agaaccacca gagtcacccc caagctgtga 30
<210> 8
<211> 89
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 8
gactctggtg gttctgaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat 60
cctggcccaa ctaatctgtc agatattat 89
<210> 9
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 9
cgaggctgat cagcgggttt aaactcaatg gtgatggtga 40
<210> 10
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 10
cagcgggttt aaacttagac tttcctcttc ttcttggg 38
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 11
accggaatcc cttctgcagc acc 23
<210> 12
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 12
aaacggtgct gcagaaggga ttc 23
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 13
cacagtatgt ctctggcgtt 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 14
tctcttggag tgtctcctca 20
<210> 15
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 15
accggtcatc ttagtcatta cctg 24
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 16
aaaccaggta atgactaaga tgac 24
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 17
aggaggactt gcccaacttt 20
<210> 18
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>引物
<400> 18
ctgtgtcaga acatgctgga a 21
<210> 19
<211> 1482
<212> DNA
<213> Artificial Sequence
<220>
<223>化学合成的PCR模板DNA
<400> 19
gctagcgccg gagagcttca aaaggggaac gaactcgcac taccgtctaa atacgtgaat 60
ttcctgtatt tagcgtccca ttacgagaag ttgaaaggtt cacctgaaga taacgaacag 120
aagcaacttt ttgttgagca gcacaaacat tatctcgacg aaatcataga gcaaatttcg 180
gaattcagta agagagtcat cctagctgat gccaatctgg acaaagtatt aagcgcatac 240
aacaagcaca gggataaacc catacgtgag caggcggaaa atattatcca tttgtttact 300
cttaccaacc tcggcgctcc agccgcattc aagtattttg acacaacgat agatcgcaaa 360
cgatacactt ctaccaagga ggtgctagac gcgacactga ttcaccaatc catcacggga 420
ttatatgaaa ctcggataga tttgtcacag cttgggggtg actctggtgg ttctcccaag 480
aagaagagga aagtcgaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat 540
cctggcccaa ctaatctgtc agatattatt gaaaaggaga ccggtaagca actggttatc 600
caggaatcca tcctcatgct cccagaggag gtggaagaag tcattgggaa caagccggaa 660
agcgatatac tcgtgcacac cgcctacgac gagagcaccg acgagaatgt catgcttctg 720
actagcgacg cccctgaata caagccttgg gctctggtca tacaggatag caacggtgag 780
aacaagatta agatgctccc caagaagaag aggaaagtcg ctactaactt cagcctgctg 840
aagcaggctg gagacgtgga ggagaaccct ggacctacca acctgtccga tatcattgag 900
aaagagaccg gcaaacagct ggtgatccag gagagcatcc tgatgctgcc cgaagaggtg 960
gaggaagtga tcggcaacaa gcccgagtcc gacatcctgg tgcacacagc ctatgatgaa 1020
tccaccgacg agaacgtgat gctgctgacc tccgatgctc ccgagtataa accctgggca 1080
ctggtgatcc aggactctaa tggagagaac aagatcaaga tgctgcccaa gaagaagagg 1140
aaagtcgaag gacggggctc cctcctgacc tgtggcgatg tggaagagaa ccccggcccc 1200
acaaacctca gtgacattat cgagaaggaa acaggaaaac agctcgtcat tcaagaatct 1260
attcttatgt tgcctgagga agtcgaagag gttattggca ataaacctga atctgatatt 1320
cttgtccata ccgcttacga tgagtccaca gatgaaaatg ttatgctgct cacatctgac 1380
gcaccagagt acaaaccatg ggcgctcgtt attcaagatt ccaacggcga aaacaaaatc 1440
aaaatgcttt ctggtggttc tcctaaaaaa aaaagaaagg tg 1482
<210> 20
<211> 5711
<212> DNA
<213> Artificial Sequence
<220>
<223> UGI表达载体pUGI-NLS
<400> 20
actcgagcgg ccgccactgt gctggatatc tgcagaattc atgactaatc tgtcagatat 60
tattgaaaag gagaccggta agcaactggt tatccaggaa tccatcctca tgctcccaga 120
ggaggtggaa gaagtcattg ggaacaagcc ggaaagcgat atactcgtgc acaccgccta 180
cgacgagagc accgacgaga atgtcatgct tctgactagc gacgcccctg aatacaagcc 240
ttgggctctg gtcatacagg atagcaacgg tgagaacaag attaagatgc tccccaagaa 300
gaagaggaaa gtctagggat ccgagctcgg taccaagctt aagtttaaac cgctgatcag 360
cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 420
tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 480
attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 540
aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg 600
cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa 660
gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 720
ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 780
ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 840
aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 900
gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 960
cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 1020
attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt 1080
gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 1140
gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 1200
tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 1260
cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 1320
tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg 1380
cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg 1440
atctgatcaa gagacaggat gaggatcgtt tcgcatggaa gcgggtaggc ctttggggca 1500
gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt 1560
ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg tcctccggag 1620
gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc tcttcctcat 1680
ctccgggcct ttcgacctgc agcccaagct taccatgacc gagtacaagc ccacggtgcg 1740
cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga 1800
ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct 1860
gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga 1920
cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc 1980
cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat 2040
ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg 2100
cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga 2160
ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc 2220
cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg 2280
cacctggtgc atgacccgca agcccggtgc ctgagcggga ctctggggtt cgaaatgacc 2340
gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa 2400
aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat 2460
ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa 2520
taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 2580
ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag 2640
agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 2700
ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 2760
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 2820
cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 2880
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 2940
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3000
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3060
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3120
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3180
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3240
gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 3300
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 3360
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 3420
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 3480
aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 3540
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtttt 3600
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3660
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3720
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3780
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 3840
cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 3900
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 3960
ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 4020
agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 4080
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 4140
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 4200
cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 4260
gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 4320
tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 4380
tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 4440
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 4500
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 4560
cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 4620
aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 4680
ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 4740
tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 4800
ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa 4860
tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg 4920
ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca 4980
tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata 5040
cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 5100
atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 5160
cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 5220
tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 5280
tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 5340
ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 5400
acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 5460
gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 5520
tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 5580
cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 5640
ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 5700
aagctggcta g 5711
<210> 21
<211> 8532
<212> DNA
<213> Artificial Sequence
<220>
<223> BE3表达载体pCMV-BE3
<400> 21
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420
actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480
gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540
gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600
aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660
acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720
tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780
ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 840
actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900
gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960
atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020
ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080
gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140
agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 1200
ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 1260
cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 1320
gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 1380
tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 1440
ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 1500
aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 1560
aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 1620
atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 1680
gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 1740
ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 1800
cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 1860
cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 1920
gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 1980
caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 2040
ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 2100
atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 2160
cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 2220
ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 2280
gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 2340
aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 2400
gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 2460
gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 2520
cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 2580
gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 2640
aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 2700
tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 2760
agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 2820
gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 2880
tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 2940
attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 3000
ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 3060
cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 3120
cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 3180
gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 3240
tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 3300
cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 3360
gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 3420
atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 3480
atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 3540
gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 3600
gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 3660
attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 3720
gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 3780
aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 3840
actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 3900
ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 3960
acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 4020
aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 4080
taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 4140
tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 4200
atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 4260
aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 4320
cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 4380
gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 4440
cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 4500
gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 4560
tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 4620
aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 4680
ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 4740
tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 4800
caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 4860
cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 4920
cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 4980
atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 5040
cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 5100
ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 5160
gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 5220
gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa 5280
aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340
gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400
agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460
ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520
aagaagaaga ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct 5580
gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 5640
cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 5700
catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 5760
agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 5820
ctgaggcgga aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt 5880
aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 5940
tacgagccgg aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat 6000
taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 6060
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 6120
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 6180
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 6240
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 6300
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 6360
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 6420
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 6480
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 6540
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 6600
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 6660
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 6720
acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 6780
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 6840
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6900
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6960
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7020
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7080
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7140
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7200
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7260
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7320
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7380
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7440
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7500
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7560
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7620
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7680
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7740
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7800
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7860
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7920
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7980
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8040
acgtcgacgg atcgggagat cgatctcccg atcccctagg gtcgactctc agtacaatct 8100
gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg 8160
agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga 8220
agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc 8280
gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8340
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8400
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8460
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8520
atcaagtgta tc 8532
<210> 22
<211> 8848
<212> DNA
<213> Artificial Sequence
<220>
<223> eBEa表达载体pCMV-eBEa
<400> 22
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420
actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480
gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540
gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600
aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660
acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720
tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780
ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 840
actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900
gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960
atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020
ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080
gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140
agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 1200
ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 1260
cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 1320
gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 1380
tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 1440
ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 1500
aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 1560
aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 1620
atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 1680
gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 1740
ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 1800
cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 1860
cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 1920
gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 1980
caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 2040
ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 2100
atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 2160
cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 2220
ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 2280
gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 2340
aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 2400
gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 2460
gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 2520
cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 2580
gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 2640
aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 2700
tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 2760
agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 2820
gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 2880
tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 2940
attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 3000
ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 3060
cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 3120
cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 3180
gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 3240
tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 3300
cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 3360
gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 3420
atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 3480
atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 3540
gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 3600
gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 3660
attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 3720
gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 3780
aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 3840
actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 3900
ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 3960
acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 4020
aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 4080
taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 4140
tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 4200
atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 4260
aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 4320
cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 4380
gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 4440
cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 4500
gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 4560
tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 4620
aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 4680
ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 4740
tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 4800
caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 4860
cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 4920
cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 4980
atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 5040
cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 5100
ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 5160
gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 5220
gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa 5280
aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340
gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400
agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460
ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520
aagaagaaga ggaaagtcgg gcccgagggc agaggaagtc tgctaacatg cggtgacgtc 5580
gaggagaatc ctggcccaac taatctgtca gatattattg aaaaggagac cggtaagcaa 5640
ctggttatcc aggaatccat cctcatgctc ccagaggagg tggaagaagt cattgggaac 5700
aagccggaaa gcgatatact cgtgcacacc gcctacgacg agagcaccga cgagaatgtc 5760
atgcttctga ctagcgacgc ccctgaatac aagccttggg ctctggtcat acaggatagc 5820
aacggtgaga acaagattaa gatgctctct ggtggttctc ccaagaagaa gaggaaagtc 5880
taagtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 5940
tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 6000
taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 6060
gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 6120
gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgatacc gtcgacctct 6180
agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 6240
acaattccac acaacatacg agccggaagc ataaagtgta aagcctaggg tgcctaatga 6300
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 6360
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 6420
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 6480
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 6540
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 6600
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 6660
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 6720
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 6780
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 6840
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 6900
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 6960
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 7020
tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 7080
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 7140
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 7200
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 7260
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 7320
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 7380
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 7440
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 7500
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 7560
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 7620
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 7680
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 7740
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 7800
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 7860
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 7920
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 7980
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 8040
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 8100
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 8160
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 8220
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 8280
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 8340
cgaaaagtgc cacctgacgt cgacggatcg ggagatcgat ctcccgatcc cctagggtcg 8400
actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt 8460
gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg 8520
accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta 8580
cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg 8640
gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc 8700
ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc 8760
atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact 8820
gcccacttgg cagtacatca agtgtatc 8848
<210> 23
<211> 9499
<212> DNA
<213> Artificial Sequence
<220>
<223> eBEb表达载体pCMV-eBEb
<400> 23
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag 420
actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 480
gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 540
gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 600
aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 660
acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 720
tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 780
ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 840
actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 900
gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 960
atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 1020
ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 1080
gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacacccgaa 1140
agtgataaaa agtattctat tggtttagcc atcggcacta attccgttgg atgggctgtc 1200
ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa cacagaccgt 1260
cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga aacggcagag 1320
gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa ccgaatatgt 1380
tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt ctttcaccgt 1440
ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc catctttgga 1500
aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca cctcagaaaa 1560
aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc tcttgcccat 1620
atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga caactcggat 1680
gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga agagaaccct 1740
ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc taaatcccga 1800
cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt gttcggtaac 1860
cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga cttagctgaa 1920
gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa tctactggca 1980
caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag cgatgcaatc 2040
ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt atccgcttca 2100
atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc cctagtccgt 2160
cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa cgggtacgca 2220
ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa acccatatta 2280
gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga tctactgcga 2340
aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg cgaattgcat 2400
gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg tgaaaagatt 2460
gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg agggaactct 2520
cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccatggaa ttttgaggaa 2580
gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa ctttgacaag 2640
aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta tttcacagtg 2700
tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc cgcctttcta 2760
agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg caaagtgaca 2820
gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc tgtcgagatc 2880
tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct cctaaagata 2940
attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga agatatagtg 3000
ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa aacatacgct 3060
cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac gggctgggga 3120
cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa aactattctc 3180
gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat ccatgatgac 3240
tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg ggactcattg 3300
cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat actccagaca 3360
gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga aaacattgta 3420
atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg 3480
atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa ggagcatcct 3540
gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca aaatggaagg 3600
gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga cgtcgatcac 3660
attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct tacacgctcg 3720
gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag 3780
aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt cgataactta 3840
actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat taaacgtcag 3900
ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc ccgaatgaat 3960
acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac tttaaagtca 4020
aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga gataaataac 4080
taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact cattaagaaa 4140
tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga cgtccgtaag 4200
atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt cttttattct 4260
aacattatga atttctttaa gacggaaatc actctggcaa acggagagat acgcaaacga 4320
cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg ccgggacttc 4380
gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa aactgaggtg 4440
cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga taagctcatc 4500
gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc tacagttgcc 4560
tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact gaagtcagtc 4620
aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa ccccatcgac 4680
ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa actaccaaag 4740
tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc cggagagctt 4800
caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta tttagcgtcc 4860
cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact ttttgttgag 4920
cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag taagagagtc 4980
atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca cagggataaa 5040
cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa cctcggcgct 5100
ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac ttctaccaag 5160
gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga aactcggata 5220
gatttgtcac agcttggggg tgactctggt ggttctacta atctgtcaga tattattgaa 5280
aaggagaccg gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg 5340
gaagaagtca ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag 5400
agcaccgacg agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct 5460
ctggtcatac aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc 5520
aagaagaaga ggaaagtcgg gcccgagggc agaggaagtc tgctaacatg cggtgacgtc 5580
gaggagaatc ctggcccaac taatctgtca gatattattg aaaaggagac cggtaagcaa 5640
ctggttatcc aggaatccat cctcatgctc ccagaggagg tggaagaagt cattgggaac 5700
aagccggaaa gcgatatact cgtgcacacc gcctacgacg agagcaccga cgagaatgtc 5760
atgcttctga ctagcgacgc ccctgaatac aagccttggg ctctggtcat acaggatagc 5820
aacggtgaga acaagattaa gatgctcccc aagaagaaga ggaaagtcgc tactaacttc 5880
agcctgctga agcaggctgg agacgtggag gagaaccctg gacctaccaa cctgtccgat 5940
atcattgaga aagagaccgg caaacagctg gtgatccagg agagcatcct gatgctgccc 6000
gaagaggtgg aggaagtgat cggcaacaag cccgagtccg acatcctggt gcacacagcc 6060
tatgatgaat ccaccgacga gaacgtgatg ctgctgacct ccgatgctcc cgagtataaa 6120
ccctgggcac tggtgatcca ggactctaat ggagagaaca agatcaagat gctgcccaag 6180
aagaagagga aagtcgaagg acggggctcc ctcctgacct gtggcgatgt ggaagagaac 6240
cccggcccca caaacctcag tgacattatc gagaaggaaa caggaaaaca gctcgtcatt 6300
caagaatcta ttcttatgtt gcctgaggaa gtcgaagagg ttattggcaa taaacctgaa 6360
tctgatattc ttgtccatac cgcttacgat gagtccacag atgaaaatgt tatgctgctc 6420
acatctgacg caccagagta caaaccatgg gcgctcgtta ttcaagattc caacggcgaa 6480
aacaaaatca aaatgctttc tggtggttct cctaaaaaaa aaagaaaggt gtgagtttaa 6540
acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 6600
cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 6660
gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 6720
gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct 6780
atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc 6840
ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 6900
cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa 6960
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 7020
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 7080
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 7140
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 7200
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 7260
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 7320
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 7380
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 7440
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 7500
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 7560
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 7620
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 7680
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 7740
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 7800
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 7860
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 7920
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 7980
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 8040
cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 8100
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 8160
ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 8220
agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 8280
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 8340
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 8400
cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 8460
gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 8520
tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 8580
tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 8640
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 8700
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 8760
cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 8820
aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 8880
ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 8940
tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 9000
ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt 9060
acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag 9120
gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat 9180
tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga 9240
tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 9300
gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 9360
tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 9420
ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 9480
gcagtacatc aagtgtatc 9499
<210> 24
<211> 4949
<212> DNA
<213> Artificial Sequence
<220>
<223> sgFANCF表达载体
<400> 24
ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60
cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120
ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180
cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240
tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300
tgtggaaagg acgaaacacc ggaatccctt ctgcagcacc gttttagagc tagaaatagc 360
aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 420
tttaaagaat tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa 480
ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata 540
caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg 600
gacagcagag atccactttg gccgcggctc gagggggttg gggttgcgcc ttttccaagg 660
cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg 720
cgccgaccct gggactcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc 780
cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc 840
cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct 900
cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa 960
tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc 1020
ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa 1080
gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc 1140
ccagggggat ccaccggagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac 1200
ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc 1260
cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact 1320
cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc 1380
ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg 1440
cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct 1500
cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc 1560
cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga 1620
gcgcgccggg gtgcccgcct tcctggaaac ctccgcgccc cgcaacctcc ccttctacga 1680
gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg 1740
catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag 1800
gagcgcacga ccccatgcat cggtaccttt aagaccaatg acttacaagg cagctgtaga 1860
tcttagccac tttctagagt cggggcggcc ggccgcttcg agcagacatg ataagataca 1920
ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 1980
tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca 2040
acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca 2100
agtaaaacct ctacaaatgt ggtaaaatcg ataaggatcc gtcgaccgat gcccttgaga 2160
gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt 2220
atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc 2280
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 2340
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 2400
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 2460
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 2520
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 2580
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 2640
ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 2700
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 2760
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 2820
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 2880
ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 2940
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 3000
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 3060
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 3120
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 3180
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 3240
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 3300
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gggacccacg 3360
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 3420
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 3480
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 3540
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 3600
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 3660
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 3720
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 3780
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 3840
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 3900
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 3960
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 4020
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 4080
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 4140
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 4200
tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 4260
cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 4320
cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 4380
tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 4440
gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 4500
tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 4560
ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 4620
taacgcgaat tttaacaaaa tattaacgtt tacaatttcc cattcgccat tcaggctgcg 4680
caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc 4740
atgataagta agtaatatta aggtacggga ggtacttgga gcggccgcaa taaaatatct 4800
ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgatagtact aacatacgct 4860
ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt 4920
gcaggtgcca gaacatttct ctatcgata 4949
<210> 25
<211> 4950
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNF2表达载体
<400> 25
ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60
cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120
ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180
cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240
tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300
tgtggaaagg acgaaacacc ggtcatctta gtcattacct ggttttagag ctagaaatag 360
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 420
ttttaaagaa ttctcgacct cgagacaaat ggcagtattc atccacaatt ttaaaagaaa 480
aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat 540
acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag 600
ggacagcaga gatccacttt ggccgcggct cgagggggtt ggggttgcgc cttttccaag 660
gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg 720
gcgccgaccc tgggactcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg 780
ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt 840
ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc actagtaccc 900
tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca 960
atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg 1020
cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca 1080
agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc 1140
cccaggggga tccaccggag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca 1200
cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg 1260
ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac 1320
tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg 1380
cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg 1440
gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc 1500
tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc 1560
ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg 1620
agcgcgccgg ggtgcccgcc ttcctggaaa cctccgcgcc ccgcaacctc cccttctacg 1680
agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt 1740
gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc ccgaccgaaa 1800
ggagcgcacg accccatgca tcggtacctt taagaccaat gacttacaag gcagctgtag 1860
atcttagcca ctttctagag tcggggcggc cggccgcttc gagcagacat gataagatac 1920
attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa 1980
atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac 2040
aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc 2100
aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag 2160
agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact 2220
tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt 2280
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 2340
caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 2400
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 2460
ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 2520
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 2580
ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 2640
tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 2700
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 2760
ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 2820
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 2880
gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 2940
aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 3000
tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 3060
ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 3120
tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 3180
aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 3240
tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 3300
ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgggacccac 3360
gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 3420
gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 3480
taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 3540
tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 3600
ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 3660
tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 3720
ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 3780
tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 3840
ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 3900
aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 3960
actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 4020
aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 4080
tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 4140
aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 4200
ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4260
ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 4320
ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4380
ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 4440
ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4500
gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4560
tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4620
ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc ccattcgcca ttcaggctgc 4680
gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag cccaagctac 4740
catgataagt aagtaatatt aaggtacggg aggtacttgg agcggccgca ataaaatatc 4800
tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc 4860
tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag 4920
tgcaggtgcc agaacatttc tctatcgata 4950
Claims (10)
1.一种碱基编辑系统，其特征在于，包括UGI表达载体或其转录产物和BE3表达载体或其转录产物，或包括UGI和BE3共表达载体或其转录产物。
2.如权利要求1所述的碱基编辑系统，其特征在于，所述的UGI和BE3共表达载体为增强型CRISPR碱基编辑器eBEa表达载体，其序列包括SEQ ID NO：22。
3.如权利要求1所述的碱基编辑系统，其特征在于，所述的UGI和BE3共表达载体为增强型CRISPR碱基编辑器eBEb表达载体，其序列包括SEQ ID NO：23。
4.如权利要求1所述的碱基编辑系统，其特征在于，所述的UGI表达载体的序列包括SEQID NO：20。
5.如权利要求1所述的碱基编辑系统，其特征在于，所述的碱基编辑系统还包括针对某物种的基因组靶点的sgRNA表达载体。
6.如权利要求1所述的碱基编辑系统，其特征在于，所述的碱基编辑系统还包括真核细胞和基因转导试剂。
7.权利要求1-6中任一项所述的碱基编辑系统的构建方法，其特征在于，包括扩增UGI基因，并连入真核表达载体中，得到UGI表达载体；或利用PCR方法扩增T2A-UGI编码序列，并同框连入BE3编码区的下游，得到到增强型CRISPR碱基编辑器eBEa表达载体；或人工合成3×T2A-UGI编码序列，PCR扩增后同框连入BE3编码区的下游，得到到增强型CRISPR碱基编辑器eBEb表达载体。
8.权利要求1-6中任一项碱基编辑系统的应用方法，其特征在于，包括构建针对某物种的基因组DNA靶点的sgRNA；在真核细胞中共引入sgRNA，BE3表达载体或其转录产物以及UGI表达载体或其转录产物，或者，在真核细胞中共引入sgRNA以及UGI和BE3共表达载体或其转录产物，使其在靶点基因组DNA发生C-T碱基编辑。
9.如权利要求8所述的碱基编辑系统的应用方法，其特征在于，还包括：对处理后的真核细胞基因组DNA进行抽提，然后利用针对靶点的特异性引物进行PCR扩增，利用基因组DNA的PCR产物构建高通量二代DNA测序文库，并进行高通量二代DNA测序。
10.如权利要求8所述的碱基编辑系统的应用方法，其特征在于，所述的某物种的基因组DNA靶点包括但不限于人的FANCF和RNF2靶点中的至少一个。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710239348.XA CN106916852B (zh) | 2017-04-13 | 2017-04-13 | 一种碱基编辑系统及其构建和应用方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710239348.XA CN106916852B (zh) | 2017-04-13 | 2017-04-13 | 一种碱基编辑系统及其构建和应用方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106916852A true CN106916852A (zh) | 2017-07-04 |
| CN106916852B CN106916852B (zh) | 2020-12-04 |
Family
ID=59568124
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710239348.XA Active CN106916852B (zh) | 2017-04-13 | 2017-04-13 | 一种碱基编辑系统及其构建和应用方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106916852B (zh) |
Cited By (35)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| CN108753823A (zh) * | 2018-06-20 | 2018-11-06 | 李广磊 | 利用碱基编辑技术实现基因敲除的方法及其应用 |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| CN109321584A (zh) * | 2017-12-27 | 2019-02-12 | 华东师范大学 | 一种简单定性/定量检测单碱基基因编辑技术工作效率的报告系统 |
| WO2019042284A1 (en) * | 2017-09-01 | 2019-03-07 | Shanghaitech University | FUSION PROTEINS FOR ENHANCED PRECISION IN THE BASIC EDITION |
| CN109576267A (zh) * | 2018-09-21 | 2019-04-05 | 中山大学 | 一种用于单碱基编辑的gRNA、载体、细胞及其制备方法 |
| CN109666693A (zh) * | 2018-12-29 | 2019-04-23 | 北京市农林科学院 | Mg132在碱基编辑系统编辑受体基因组中的应用 |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| CN110066824A (zh) * | 2018-01-24 | 2019-07-30 | 中国农业科学院植物保护研究所 | 一套用于水稻的碱基编辑人工系统 |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| WO2019227640A1 (zh) * | 2018-06-01 | 2019-12-05 | 上海科技大学 | 利用碱基编辑修复fbn1t7498c突变的试剂和方法 |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| CN111118060A (zh) * | 2020-01-16 | 2020-05-08 | 扬州大学 | 基于基因编辑的BnALS1突变型基因、蛋白及其应用 |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| CN111801345A (zh) * | 2017-07-28 | 2020-10-20 | 哈佛大学的校长及成员们 | 使用噬菌体辅助连续进化(pace)的进化碱基编辑器的方法和组合物 |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| CN112534054A (zh) * | 2018-05-11 | 2021-03-19 | 比姆医疗股份有限公司 | 使用可编程碱基编辑器系统取代病原性氨基酸的方法 |
| CN112601816A (zh) * | 2018-05-11 | 2021-04-02 | 比姆医疗股份有限公司 | 使用可编程碱基编辑器系統遏止病原性突变的方法 |
| CN112867792A (zh) * | 2018-08-23 | 2021-05-28 | 桑格摩生物治疗股份有限公司 | 工程化靶特异性碱基编辑器 |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| WO2024109745A1 (en) * | 2022-11-21 | 2024-05-30 | CorrectSequence Therapeutics Co., Ltd | Gene editing systems and methods for treating hbv infection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016070129A1 (en) * | 2014-10-30 | 2016-05-06 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
-
2017
- 2017-04-13 CN CN201710239348.XA patent/CN106916852B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016070129A1 (en) * | 2014-10-30 | 2016-05-06 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
Non-Patent Citations (5)
| Title |
|---|
| ALEXIS C. KOMOR ET AL.: "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage", 《NATURE》 * |
| APRIL J. SCHUMACHER ET AL.: "APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast", 《PNAS》 * |
| KOMOR ET AL.: "Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity", 《SCI. ADV.》 * |
| LAURENCE H. PEARL: "Structure and function in the uracil-DNA glycosylase superfamily", 《MUTATION RESEARCH》 * |
| LIJIE WANG ET AL.: "Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor", 《CELL RESEARCH》 * |
Cited By (61)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US12006520B2 (en) | 2011-07-22 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US12043852B2 (en) | 2015-10-23 | 2024-07-23 | President And Fellows Of Harvard College | Evolved Cas9 proteins for gene editing |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11999947B2 (en) | 2016-08-03 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US12084663B2 (en) | 2016-08-24 | 2024-09-10 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| CN111801345A (zh) * | 2017-07-28 | 2020-10-20 | 哈佛大学的校长及成员们 | 使用噬菌体辅助连续进化(pace)的进化碱基编辑器的方法和组合物 |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| CN111065647B (zh) * | 2017-09-01 | 2024-06-11 | 上海科技大学 | 用于提高碱基编辑精度的融合蛋白 |
| WO2019042284A1 (en) * | 2017-09-01 | 2019-03-07 | Shanghaitech University | FUSION PROTEINS FOR ENHANCED PRECISION IN THE BASIC EDITION |
| CN111065647A (zh) * | 2017-09-01 | 2020-04-24 | 上海科技大学 | 用于提高碱基编辑精度的融合蛋白 |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| CN109321584A (zh) * | 2017-12-27 | 2019-02-12 | 华东师范大学 | 一种简单定性/定量检测单碱基基因编辑技术工作效率的报告系统 |
| CN109321584B (zh) * | 2017-12-27 | 2021-07-16 | 华东师范大学 | 一种简单定性/定量检测单碱基基因编辑技术工作效率的报告系统 |
| CN110066824A (zh) * | 2018-01-24 | 2019-07-30 | 中国农业科学院植物保护研究所 | 一套用于水稻的碱基编辑人工系统 |
| CN110066824B (zh) * | 2018-01-24 | 2021-06-08 | 中国农业科学院植物保护研究所 | 一套用于水稻的碱基编辑人工系统 |
| CN112601816A (zh) * | 2018-05-11 | 2021-04-02 | 比姆医疗股份有限公司 | 使用可编程碱基编辑器系統遏止病原性突变的方法 |
| CN112534054A (zh) * | 2018-05-11 | 2021-03-19 | 比姆医疗股份有限公司 | 使用可编程碱基编辑器系统取代病原性氨基酸的方法 |
| WO2019227640A1 (zh) * | 2018-06-01 | 2019-12-05 | 上海科技大学 | 利用碱基编辑修复fbn1t7498c突变的试剂和方法 |
| CN108753823B (zh) * | 2018-06-20 | 2022-09-23 | 李广磊 | 利用碱基编辑技术实现基因敲除的方法及其应用 |
| CN108753823A (zh) * | 2018-06-20 | 2018-11-06 | 李广磊 | 利用碱基编辑技术实现基因敲除的方法及其应用 |
| CN112867792A (zh) * | 2018-08-23 | 2021-05-28 | 桑格摩生物治疗股份有限公司 | 工程化靶特异性碱基编辑器 |
| CN109576267A (zh) * | 2018-09-21 | 2019-04-05 | 中山大学 | 一种用于单碱基编辑的gRNA、载体、细胞及其制备方法 |
| CN109666693A (zh) * | 2018-12-29 | 2019-04-23 | 北京市农林科学院 | Mg132在碱基编辑系统编辑受体基因组中的应用 |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN111118060A (zh) * | 2020-01-16 | 2020-05-08 | 扬州大学 | 基于基因编辑的BnALS1突变型基因、蛋白及其应用 |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US12031126B2 (en) | 2020-05-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| WO2024109745A1 (en) * | 2022-11-21 | 2024-05-30 | CorrectSequence Therapeutics Co., Ltd | Gene editing systems and methods for treating hbv infection |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106916852B (zh) | 2020-12-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106916852B (zh) | 一种碱基编辑系统及其构建和应用方法 | |
| KR102319845B1 (ko) | 조류 숙주 세포에 대한 crispr-cas 시스템 | |
| KR100880509B1 (ko) | 재조합 단백질의 대량 생산을 위한 신규한 벡터, 발현세포주 및 이를 이용한 재조합 단백질의 생산 방법 | |
| CN113272435A (zh) | 在多个主要组织相容性复合体分子上受限的ny-eso-1特异性t细胞受体的组合物 | |
| CN111172133B (zh) | 一种碱基编辑工具及其用途 | |
| CN108753778B (zh) | 利用碱基编辑修复fbn1t7498c突变的试剂和方法 | |
| KR20180048743A (ko) | 2a 펩타이드를 포함하는 재조합 벡터 | |
| CN111836825A (zh) | 优化的植物crispr/cpf1系统 | |
| WO2004067743A1 (en) | Vaccine production using transposon based vectors | |
| CN110072546B (zh) | 新的猪流感疫苗 | |
| AU2016378480A1 (en) | Endothelium-specific nucleic acid regulatory elements and methods and use thereof | |
| KR20200022486A (ko) | 조작되고 완전-기능 맞춤 당단백질 | |
| KR20210108423A (ko) | 아데노 관련 바이러스 (aav) 생산자 세포주 및 관련 방법 | |
| KR102618843B1 (ko) | 신규한 ehv 삽입 부위 orf70 | |
| KR20210105382A (ko) | 단백질을 코딩하는 rna | |
| US20040235011A1 (en) | Production of multimeric proteins | |
| CN111094569A (zh) | 光控性病毒蛋白质、其基因及包含该基因的病毒载体 | |
| CN115927299A (zh) | 增加双链rna产生的方法和组合物 | |
| KR102543774B1 (ko) | 신규한 프로모터 | |
| CN111088176B (zh) | 产β-胡萝卜素的基因工程菌及其应用 | |
| CN112063655A (zh) | 一种端粒基因治疗产品和哺乳动物广泛性启动子的用途 | |
| CN116135974A (zh) | 一种重组糖基化酶碱基编辑系统及其应用 | |
| CN112322655B (zh) | 一种不受基因序列限制的碱基编辑系统及其制备方法和应用 | |
| KR20230072150A (ko) | 강력한 적응성 면역반응 유도 및 모체이행항체의 간섭을 극복하는 재조합 구제역 a형 바이러스 및 이를 포함하는 구제역 백신 조성물 | |
| KR20230072149A (ko) | 강력한 적응성 면역반응 유도 및 모체이행항체의 간섭을 극복하는 재조합 구제역 o형 바이러스 및 이를 포함하는 구제역 백신 조성물 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |