CN114836411A - 基于crispr技术的靶基因捕获的方法 - Google Patents
基于crispr技术的靶基因捕获的方法 Download PDFInfo
- Publication number
- CN114836411A CN114836411A CN202210367292.7A CN202210367292A CN114836411A CN 114836411 A CN114836411 A CN 114836411A CN 202210367292 A CN202210367292 A CN 202210367292A CN 114836411 A CN114836411 A CN 114836411A
- Authority
- CN
- China
- Prior art keywords
- artifical sequence
- dna
- sequence
- rna
- target gene
- 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.)
- Pending
Links
Images
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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Crystallography & Structural Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种基于CRISPR技术的新型靶基因捕获的方法(CPCS)，其特征在于：使用sgRNA Cas蛋白复合体进行对靶基因的序列切割捕获，同时使用核酸水解酶消解非靶基因序列得到靶基因样本。本发明使用CRISPR技术结合核酸水解酶进行靶基因捕获，利用CRISPR的进行精准定位并切割的特点进行靶基因序列与非靶基因序列分开，同时利用Cas蛋白切割后保留在切割位点的特点使用核酸水解酶对非靶基因进行水解从而获得靶基因。本发明具有低成本，高特异性，短时长捕获的特点，最短仅需5小时即可完成靶基因捕获建库流程。
Description
技术领域
本申请涉及分子生物学领域，具体涉及一种基于CRISPR技术的靶基因捕获的方法。
背景技术
随着二代测序(Next-generation sequencing，NGS)技术高速发展，测序技术出现了两个分支：1)全基因组测序；2)靶向捕获测序。
由于全基因组测序可获得某一物种的个体或群体核DNA的全部遗传信息，于参考基因组对比可获得非常全面的遗传标记信息，同时利用二代测序的技术可获得未知生物的序列，进而在科研、临床感染、物种进化等领域上具有广泛的应用。但面临单个或多个基因的精准定位研究时，由于在庞大的遗传信息中所需要的仅占0.0001％～2％，占比极低，极大部分均为无效数据，尽管测序数据有极少量的所需信息，但由于数据量过少导致无法得到准确的判断；由于在单个或多个基因的精准定位研究中无效数据过多，导致单个样本测序成本过高，迫使研究人员开发另一种方法：靶向捕获测序。
将DNA靶向捕获测序技术应用到二代测序中，可将目标基因富集，提高目标基因在文库中的占比，以及大幅度提高测序深度。例如，外显子在全基因组中仅占1％～2％，使用IDT(Integrated DNA Technologies)的全外显子捕获时，可将外显子的测序数据捕获至90％以上；检测极限最低可至0.1％。相较于非捕获的全基因组测序，成本下降约100倍。因此，靶向捕获测序成为遗传病、肿瘤检测、位点突变研究等领域上具有广泛的应用；高通量测序可直接获得大量相关位点变异信息，对于精准医疗也具有重大意义。但常规基于靶向捕获的方法主要有固相载体芯片与固相载体磁珠的技术。均需要对固相载体进行修饰后，才能进行特异性结合。例如M-270磁珠，对磁珠与探针进行亲和霉素修饰，探针特异性结合靶序列，磁珠特异性结合探针；需要长时间的过夜杂交。时间长，约20小时；且成本仍然过高，约240元每毫克。
随着CRISPR技术研究越发深入，其功能也越发完整。CRISPR技术大都用于插入或定点沉默激活等用途。基于CRISPR技术的靶向测序技术研究仍处于基础研究阶段。
发明内容
本发明的目的是提供一种新型靶基因捕获的方法(CPCS)及试剂，我们将其命名为：CRISPR-Protect Capture Sequence(CPCS)。
本发明采取的技术方案为：一种基于CRISPR技术的靶基因捕获的方法，其特征在于其步骤包括：
(1)设计并制备靶基因的sgRNA库，将制备好的sgRNA与Cas蛋白结合，获得sgRNA/Cas复合体；
(2)将sgRNA/Cas复合体与待测样本混合孵育，获得被sgRNA/Cas复合体切割后的被处理样本；
(3)在被处理样本中加入核酸水解酶，水解非靶基因的序列，然后终止核酸水解酶反应，
纯化获得靶基因样本。
优选的，所述Cas蛋白包括但不限于Cas9、Cas12、Cas13、Cas14。
优选的，所述sgRNA库根据靶基因两端的序列进行设计，靶基因序列两端sgRNA间隔不超过10KB，也即欲富集的靶基因区域最好不要超过10KB。
优选的，步骤(1)中的结合条件为在1×Cas蛋白反应液中以20～40℃条件下结合。
优选的，所述的Cas蛋白反应液中含有Tris-HCl、NaCl、MgCl2、BSA和Tween；
所述的Tris-HCl浓度为10mM～200mM；
所述的NaCl浓度为10mM～200mM；
所述的MgCl2浓度为1mM～50mM；
所述的BSA浓度为10ng/μL～50ng/μL；
所述的Tween浓度为0.01％～2％。
优选的，步骤(2)中孵育温度为25℃～50℃。
优选的，步骤(3)中的核酸水解酶为大肠杆菌外切酶1、大肠杆菌外切酶3、大肠杆菌外切酶7、噬菌体核酸外切酶、T7噬菌体基因Ⅵ核酸外切酶或Bal 31核酸酶；
优选的，所述的核酸水解酶使用量为1U/rxn～1000U/rxn。
优选的，步骤(3)中在25℃～50℃条件下进行水解。
优选的，所述sgRNA库的制备通过以下步骤制得：
A、合成T7启动子-gRNA-Cas蛋白骨架的DNA序列；
B、使用T7启动子-gRNA-Cas蛋白骨架DNA序列的5端重叠区域为上游引物，通用Cas蛋白骨架DNA序列的3端序列作为下游引物，添加Cas蛋白骨架DNA作为模板，扩增得到T7启动子-gRNA-Cas蛋白骨架DNA；
C、使用T7体外转录获得sgRNA库。
本发明的靶基因捕获方法不但可以用于mRNA建库，也可以用于gDNA直接建库；
与现有技术相比，本发明具有如下有益效果：
(1)与传统捕获的方法相比，本技术无需在建库时加入大量的非目标片段参与建库，能最大限度的放大目标区域的基因。
(2)与传统捕获的方法相比，本技术无需使用大量带修饰的探针和昂贵的带修饰的固相载体，能最大限度的降低目标基因的捕获成本。
(3)与传统捕获的方法相比，本捕获方法可完整保留一条或多条目标基因，其目标基因的完整度能得到最大限度的保障。
(4)与传统捕获的方法相比，本技术最短可在5小时完成捕获建库完整流程，极大程度上提升了检测效率，节约时间成本。
附图说明
图1为基于CRISPR技术的捕获流程示意图。
图2为本发明的捕获原理图。
图3为gRNA oligo序列设计图。
图4为实施例2中测序样品1、2、3、4、5、6、7、8、9、10中EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET基因的占比图。
图5为实施例2中的测序样品1的EGFR基因的单条sgRNA在Target Reads中的占比图。
图6为实施例2中的测序样品2的KRAS基因的单条sgRNA在Target Reads中的占比图。
图7为实施例2中的测序样品3的BRAF基因的单条sgRNA在Target Reads中的占比图。
图8为实施例2中的测序样品4的HER2基因的单条sgRNA在Target Reads中的占比图。
图9为实施例2中的测序样品5的ALK基因的单条sgRNA在Target Reads中的占比图。
图10为实施例2中的测序样品6的ROS1基因的单条sgRNA在Target Reads中的占比图。
图11为实施例2中的测序样品7的MET基因的单条sgRNA在Target Reads中的占比图。
图12为实施例2中的测序样品8的RET基因的单条sgRNA在Target Reads中的占比图。
图13为实施例3中的测序样品与标准品实际位点突变率的测序分析对比图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例对本发明作进一步地说明。基于CRISPR技术的捕获流程如图1所示。肺癌是目前研究较多、检测方法较多、临床数据较多的疾病。故采用肺癌相关基因(EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET)进行阐述。
实施例1gRNA设计序列及sgRNA库制备
根据肺癌相关基因(EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET)序列设计下述sgRNA，所有的引物均由生工生物工程(上海)股份有限公司代为合成。
gRNA设计序列如表1所示:
根据表1所设计的gRNA oligo在合成时，在gRNA oligo前端加上T7启动子序列：5’-TTCTAATACGACTCACTATA-3’；并在gRNA oligo后端加上Cas蛋白通用骨架序列：5’-GTTTTAGAGCTAGA-3’。
最终合成gRNA oligo序列为：5’-TTCTAATACGACTCACTATA-gRNA-GTTTTAGAGCTAGA-3’，如图3所示，还需要在T7启动子序列与gRNA之间添加G，添加规则为：如果gRNA前两位是G就不用添加，如果第一位是G就添加一个G，如果第一位和第二位不是G就添加两个GG。以该序列作为上游引物，以5’-AAAAGCACCGACTCGGTGCC-3’为下游通用引物，添加少部分Cas蛋白骨架序列作为模板进行单条或多条sgRNA的扩增，Cas9骨架序列为：AATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT，得到DNA产物，以DNA产物为模板，进行sgRNA-Cas蛋白骨架RNA体外转录，转录后完整sgRNA序列如表2所示。
sgRNA库制备具体实施流程如下(sgRNA扩增采用
Precision sgRNASynthesis Kit，YEASEN)：
1)加入Scaffold Template 1.25μL；gRNA oligo(10μM)；下游通用引物(10μM)；2×Canace Enzyme Mix 12.5μL；补水至25μL；
2)98℃10S；68℃10S；30cycles；
3)取5μL进行电泳，验证条带是否单一；
sgRNA库体外转录具体实施流程如下(sgRNA库体外转录采用
T7 HighYield RNA Synthesis Kit，YEASEN)：
1)10×Transcription Buffer 2μL；CTP/GTP/ATP/UTP(100mM)各2μL；T7 RNAPolymerase Mix 2μL；sgRNA库1μg；补水至20μL；
2)37℃120min；
3)加入DNaseⅠ2μL；
4)37℃15min；
5)2.0×RNA Cleaner(Hieff
RNA Cleaner，YEASEN)磁珠纯化；
6)取5μL进行电泳，验证条带是否单一；
实施例2sgRNA测试
1.根据EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET 8个基因所设计的sgRNA分别等浓度混合为EGFR-sgRNA Mix、KRAS-sgRNA Mix、BRAF-sgRNA Mix、HER2-sgRNA Mix、ALK-sgRNA Mix、ROS1-sgRNA Mix、MET-sgRNA Mix、RET-sgRNA Mix。以KRAS-sgRNA Mix为例，比如KRAS基因中设计有4条gRNA，经过实施例1步骤后分别获得4条浓度不一致的KRAS的sgRNA文库，随后进行等量混合，即获得KRAS-sgRNA Mix。
2.实验方法：
1)使用mRNA捕获试剂盒(Hieff
mRNA Isolation Master Kit mRNA，YEASEN)制备mRNA：1)RNA模板(需要进行捕获测序的RNA样本)投入1μg；补水至50μL；加入mRNACapture Beads 50μL，吹打混匀；2)65℃，5min；25℃，5min；25℃，hold；3)室温静置5min；4)去除上清；5)使用200μL Beads Wash Buffer重悬磁珠洗涤，移除上清，重复两次；6)加入50μL Tris Buffer重悬磁珠，混匀，静置5min；7)移除上清，使用200μL Beads Wash Buffer重悬磁珠洗涤，移除上清；8)15μL DEPC-H2O重悬磁珠；80℃2min；立即转移至新PCR管。
2)使用RNA建库试剂盒(Hieff
DNA&RNA Library Co-Prep Kit forIllumina，YEASEN)制备二链CDNA：1)Dt23VN 1μL；步骤1制得的mRNA 14μL；70℃5min；立即置于冰上；2)1st Reaction Buffer 8μL；1st Strand Enzyme Mix 2μL；加热变性的RNA 15μL；25℃5min；42℃30min；85℃5min；4℃hold；3)1st Strand cDNA 25μL；2nd ReactionBuffer 7μL；2nd Strand Enzyme Mix 3μL；16℃30min；4℃hold；4)加入1.2×DNA Cleaner(Hieff
DNA Selection Beads，YEASEN)纯化；5)22μL H2O洗脱；使用Qubit测定CDNA浓度。
3)靶基因捕获：预结合：1)sgRNA Mix 1μg；Cas蛋白10pmol；10×Cas蛋白反应液3μL；补水至29μL；25～30 30min(本实施例中使用25℃)；靶序列切割：2)加入步骤2)制得的二链CDNA模板100ng；30℃～40℃60min～120min(本实施例中使用37℃60min)；非靶序列消化：3)加入核酸水解酶5U/rxn～100U/rxn 30℃～40℃60min～240min；75℃20min(本实施例中使用核酸外切酶III与T7核酸外切酶混合，每种酶10U/rxn 37℃120min)；纯化：4)加入1.0～1.8×DNA Cleaner(Hieff
DNA Selection Beads，YEASEN)纯化；5)22μL H2O洗脱。10μL用于电泳；10μL用于建库。
4)使用DNA建库试剂盒(Hieff
OnePot Pro DNA Library Prep Kit forIllumina，YEASEN)进行靶序列建库：1)
Buffer 10μL；
Enzyme 5μL；靶基因捕获DNA10μL；补水至60μL；4℃1min；30 20min；65℃20min；4℃hold；2)LigationEnhancer 30μL；Novel T4 DNA Ligase 5μL；DNA Adapter 5μL；dA-tailed DNA(1)产物)60μL；20℃15min；4℃hold；3)0.6×DNA Cleaner(Hieff
DNA Selection Beads，YEASEN)纯化；4)22μL H2O洗脱；5)
Pro Amplification Mix 25μL；纯化产物20μL；I5/I7 5μL；98℃1min；(98℃10S；60℃30S；72℃30S)×10；72℃1min；4℃hold；6)0.9×DNACleaner(Hieff
DNA Selection Beads，YEASEN)纯化；7)22μL H2O洗脱；8)使用Qubit测定浓度，并进行电泳与测序；
3.实验设计：
本实施例共10个测序样品，分别是：(1)sgRNA采用EGFR-sgRNA Mix；(2)sgRNA采用KRAS-sgRNA Mix；(3)sgRNA采用BRAF-sgRNA Mix；(4)sgRNA采用HER2-sgRNA Mix；(5)sgRNA采用ALK-sgRNA Mix；(6)sgRNA采用ROS1-sgRNA Mix；(7)sgRNA采用MET-sgRNA Mix；(8)sgRNA采用RET-sgRNA Mix；(9)sgRNA采用EGFR-sgRNA Mix、KRAS-sgRNA Mix、BRAF-sgRNAMix、HER2-sgRNA Mix、ALK-sgRNA Mix、ROS1-sgRNA Mix、MET-sgRNA Mix、RET-sgRNA Mix等浓度混合Mix；(10)不进行靶基因捕获；
4.结果分析：
1)靶向富集原理：Cas蛋白切割后蛋白保留在gRNA结合位点，具有空间位置的占据效果，核酸水解酶在水解过程中无法通过被Cas蛋白占据的位点，及Cas蛋白对该靶位点具有保护左右；靶序列上下游分别设计gRNA对靶序列进行切割保护(图2)。切割完成后使用核酸水解酶对未被保护的序列区域进行水解；随后进行失活、纯化、建库。分析结果如图4-12所示。
表3.各样本测序数据
*测序平台为illumina。
2)Mapping(％)及Target Reads(％)分析：在测序数据分析中，Mapping(％)＝MapReads/Reads*100％；Target Reads(％)＝Target Reads/Map Reads*100％。由于sgRNA的存在导致最终数据中有极小部分的sgRNA数据残留，故Mapping率相较于常规建库略低约1％～2％；相较于传统的探针法捕获的捕获效率，本方法在的捕获效率高于传统的探针法，这得益于Cas蛋白的的特异性切割与核酸水解酶的共同作用。
表4.各基因在样本中的占比如下(％)
3)基因占比(％)分析：各样本中的EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET 8个基因占比数据拆分进行分析。单个基因具有良好的特异性富集率，在面对多个基因的复合Mix时同样具有良好的特异性富集率，其靶向率并没有受靶序列的增多而减少。在多个基因的复合Mix中，单个基因的富集率受其sgRNA效率所影响；如HER2基因所设计的sgRNA具有良好的特异性及工作效率，故HER2基因无论在单基因富集还是在多个基因富集时相较于其他基因具有最高的靶向率。
表5.Target Reads中各sgRNA所占reads数
4)单条sgRNA在单个基因样本(％)的占比分析：根据EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET 8个基因中共134条sgRNA序列进行逐条序列的所在位点进行分析，分析单条sgRNA在单基因中占比，对其评定工作效率。在EGFR基因的22条sgRNA中：EGFR-4、EGFR-5、EGFR-7、EGFR-9、EGFR-10在捕获位点分析中占比远低于平均数(1/21*100％)，效率过低，可调整sgRNA序列；在KRAS基因的4条sgRNA中效率无大幅度差距，在可接受范围内，无需调整；在BRAF基因的16条sgRNA中效率无大幅度差距，在可接受范围内，无需调整；在HER2基因的10条sgRNA中效率无大幅度差距，在可接受范围内，无需调整；在ALK基因的29条sgRNA中：ALK-13在捕获位点分析中占比远于平均数(1/29*100％)，效率偏低，可适当调整sgRNA序列；在ROS1基因的44条sgRNA中ROS1-1、ROS1-43在捕获位点分析中占比低于平均数(1/44*100％)，效率偏低，可适当调整sgRNA序列；在MET基因的25条sgRNA中MET-4、MET-6、MET-13、MET-14、MET-21在捕获位点分析中占比低于平均数(1/25*100％)，效率偏低，可适当调整sgRNA序列；在RET基因的17条sgRNA中效率无大幅度差距，在可接受范围内，无需调整。
实施例3肺癌标准品捕获建库
1.根据EGFR、KRAS、BRAF、HER2、ALK、ROS1、MET、RET 8个基因所设计的sgRNA等浓度混合为EGFR-sgRNA Mix、KRAS-sgRNA Mix、BRAF-sgRNA Mix、HER2-sgRNA Mix、ALK-sgRNAMix、ROS1-sgRNA Mix、MET-sgRNA Mix、RET-sgRNA Mix。
2.实验方法：
1)靶基因捕获：预结合：1)sgRNA Mix 1μg；Cas蛋白10pmol；10×Cas蛋白反应液3μL；补水至29μL；25～30 30min(本实施例中使用25℃)；靶序列切割：2)加入肺癌标准品(菁良，GW-OGTM800)模板100ng；30℃～40℃60min～120min(本实施例中使用37℃60min)；非靶序列消化：3)加入核酸水解酶5U/rxn～100U/rxn 30℃～40℃60min～240min；75℃20min(本实施例中使用10U/rxn 37℃120min)；纯化：4)加入1.0～1.8×DNA Cleaner(Hieff
DNA Selection Beads，YEASEN)纯化；5)22μL H2O洗脱。10μL用于电泳；10μL用于建库。
2)使用DNA建库试剂盒(Hieff
OnePot Pro DNA Library Prep Kit forIllumina，YEASEN)进行靶序列建库：1)
Buffer 10μL；
Enzyme 5μL；靶基因捕获DNA 10μL；补水至60μL；4℃1min；30 20min；65℃20min；4℃hold；2)LigationEnhancer 30μL；Novel T4 DNA Ligase 5μL；DNA Adapter 5μL；dA-tailed DNA(1)产物)60μL；20℃15min；4℃hold；3)0.6×DNA Cleaner(Hieff
DNA Selection Beads，YEASEN)纯化；4)22μL H2O洗脱；5)
Pro Amplification Mix 25μL；纯化产物20μL；I5/I7 5μL；98℃1min；(98℃10S；60℃30S；72℃30S)×10；72℃1min；4℃hold；6)0.9×DNA Cleaner(Hieff
DNA Selection Beads，YEASEN)纯化；7)22μL H2O洗脱；8)使用Qubit测定浓度，并进行电泳与测序；结果如图13所示。
3.实验设计：
本实施例共1个测序样品：(1)sgRNA采用EGFR-sgRNA Mix、KRAS-sgRNA Mix、BRAF-sgRNA Mix、HER2-sgRNA Mix、ALK-sgRNA Mix、ROS1-sgRNA Mix、MET-sgRNA Mix、RET-sgRNAMix等浓度混合Mix；
4.结果分析：
表6.测序数据
*测序平台为illumina。
1)Mapping(％)及Target Reads(％)分析：在测序数据分析中，Mapping(％)＝MapReads/Reads*100％；Target Reads(％)＝Target Reads/Map Reads*100％。实际突变样本可Map率与靶向率无异常。
表7.肺癌标准品(菁良，GW-OGTM800)突变频率与实际测序突变频率
*测序平台为illumina。
2)肺癌标准品(菁良，GW-OGTM800)突变频率与实际测序突变频率分析：肺癌标准品(菁良，GW-OGTM800)的预期突变频率与本方法测试出的突变频率差距在可控范围以内。
序列表
<110> 翌圣生物科技（上海）股份有限公司
<120> 基于CRISPR技术的靶基因捕获的方法
<141> 2022-02-11
<160> 334
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 1
atcattttct cagcctccag 20
<210> 2
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 2
cctcattgcc ctcaacacag 20
<210> 3
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 3
aaatcctgca tggcgccgtg 20
<210> 4
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 4
ctgtcccaat gggagctgct 20
<210> 5
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 5
ccgggcaggt cttgacgcag 20
<210> 6
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 6
ccagatggat gtgaaccccg 20
<210> 7
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 7
gtgtgtaacg gaataggtat 20
<210> 8
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 8
ccaggcagtc gctctcccgg 20
<210> 9
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 9
gtggagatcg ccactgatgg 20
<210> 10
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 10
agttcctgtg gatccagagg 20
<210> 11
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 11
catgccttgt gctcccccga 20
<210> 12
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 12
cttgagggag cgtaatccca 20
<210> 13
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 13
tcgttcggaa gcgcacgctg 20
<210> 14
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 14
gcgttcggca cggtgtataa 20
<210> 15
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 15
ctgcgtgatg agctgcacgg 20
<210> 16
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 16
tcgcttggtg caccgcgacc 20
<210> 17
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 17
ctggcaggga ttccgtcata 20
<210> 18
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 18
ctgaatgaca aggtagcgct 20
<210> 19
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 19
aaccagcaac aattccaccg 20
<210> 20
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 20
agaagtcctg ctggtagtca 20
<210> 21
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 21
ctgctgggcg cggggttcag 20
<210> 22
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 22
ggacagtgtt gagatactcg 20
<210> 23
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 23
ctgaattagc tgtatcgtca 20
<210> 24
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 24
ggaccagtac atgaggactg 20
<210> 25
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 25
gatgtaccta tggtcctagt 20
<210> 26
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 26
aaagacaaga cagagagtgg 20
<210> 27
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 27
gggccaggct ctgttcaacg 20
<210> 28
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 28
cctattggac aaatttggtg 20
<210> 29
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 29
aacagttatt ggaatctctg 20
<210> 30
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 30
ggcaggaaga ctctaacgat 20
<210> 31
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 31
gagagaagaa accaattggt 20
<210> 32
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 32
tataaccaca tgtttgacag 20
<210> 33
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 33
gggcccaata gagtccgagg 20
<210> 34
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 34
gcgcatcagc tggttcaaag 20
<210> 35
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 35
gtgttcagag gatcaaccac 20
<210> 36
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 36
atgattggga gattcctgat 20
<210> 37
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 37
ttgaaggctt gtaactgctg 20
<210> 38
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 38
aactggctat tgttacccag 20
<210> 39
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 39
acagtgaaat ctcgatggag 20
<210> 40
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 40
ctgttgttga tgtttgaata 20
<210> 41
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 41
gcgagaattt ggggaaagag 20
<210> 42
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 42
tcgggctggt ttccaaacag 20
<210> 43
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 43
tttgtgattg aataaggaga 20
<210> 44
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 44
aagtatatgt gaggcacatg 20
<210> 45
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 45
caggcaggag tgcatatctt 20
<210> 46
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 46
gaggacaaga agatcatggg 20
<210> 47
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 47
tctcaatctt tagacactgt 20
<210> 48
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 48
tagaagggaa taaaaaggtt 20
<210> 49
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 49
acagtttttt acagatggaa 20
<210> 50
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 50
gttaagaata gaatcattct 20
<210> 51
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 51
ccaggaagct tggacgatgt 20
<210> 52
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 52
tactacctgt aagccaatcg 20
<210> 53
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 53
gagtagctga gtggctcccg 20
<210> 54
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 54
gtaggtcccg ggcgtagaca 20
<210> 55
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 55
gggcgaggag gcgatcttgg 20
<210> 56
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 56
ggtggattcg ccaaggcgaa 20
<210> 57
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 57
cgggactggt catagctcct 20
<210> 58
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 58
ctggcgccgc atcccctccg 20
<210> 59
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 59
agcacaccat cctgagtccg 20
<210> 60
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 60
gccctggaat acatctccag 20
<210> 61
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 61
atgtgccgga aactgcctgt 20
<210> 62
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 62
atggcaggtc aggaccctaa 20
<210> 63
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 63
caggctcaag ccttcatagg 20
<210> 64
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 64
tcacatggag agctcttgat 20
<210> 65
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 65
aaacaaggag ctgaaacccg 20
<210> 66
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 66
gggagcgagg gccccctgaa 20
<210> 67
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 67
gcccagcaca gacacgccgt 20
<210> 68
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 68
cccctgatca ttgcagccgg 20
<210> 69
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 69
ggggcaggaa tgtcctccgg 20
<210> 70
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 70
ggaggcggag gatatatagg 20
<210> 71
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 71
ctgcttctgt gaccacggga 20
<210> 72
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 72
gcagctcctg gtgcttccgg 20
<210> 73
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 73
atgagggtga tgtttttccg 20
<210> 74
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 74
tgatgatcag ggcttccatg 20
<210> 75
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 75
ggcgagggcg ggtctctcgg 20
<210> 76
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 76
cagggcgagc tactatagaa 20
<210> 77
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 77
ttatccggta tacaggccca 20
<210> 78
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 78
tcaacaggca aaacgggagg 20
<210> 79
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 79
agcttgtgga acccaacgta 20
<210> 80
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 80
tgcaactggg agacttccgg 20
<210> 81
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 81
cctcgtaatg accagctcca 20
<210> 82
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 82
aaagtgtcgg gagtcgtgtg 20
<210> 83
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 83
tgtgtccaga ccgtcctatg 20
<210> 84
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 84
gagctcaagt cccgacactg 20
<210> 85
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 85
gcagtaaatg aagttggtga 20
<210> 86
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 86
ggtatatagc atccaaccga 20
<210> 87
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 87
gacctgagaa ttttttacag 20
<210> 88
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 88
tgaggtgcac taatagaggg 20
<210> 89
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 89
cagcaaaggg gatgcgaggt 20
<210> 90
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 90
gcaccgaaag ctcctgcggg 20
<210> 91
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 91
ctggcagaac tggacctatg 20
<210> 92
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 92
accagggtag tacccactga 20
<210> 93
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 93
tgtgtggctg ctgaatggga 20
<210> 94
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 94
gctattggtg aatgacatgg 20
<210> 95
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 95
ctggaccaca ctctattcag 20
<210> 96
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 96
aatgcactga tgtactatag 20
<210> 97
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 97
gggttgcaga attcggtgca 20
<210> 98
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 98
ggtccccctg cggtagactg 20
<210> 99
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 99
gtcactcctt atacctactg 20
<210> 100
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 100
aagttgtggt ggaatttagg 20
<210> 101
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 101
tgtaaaggcc ctaacctgga 20
<210> 102
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 102
ccaagaagag ttatgaggtg 20
<210> 103
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 103
gatgggatat tatgctgaag 20
<210> 104
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 104
gagaggtgaa gattcacaat 20
<210> 105
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 105
agaatctgga ataaccttag 20
<210> 106
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 106
atctgggcaa tggatctgga 20
<210> 107
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 107
atactggatc atcacagcaa 20
<210> 108
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 108
agaaacgctt tatctggaaa 20
<210> 109
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 109
tggtgatgcc ataccatgtg 20
<210> 110
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 110
cctggtggta aatgttccaa 20
<210> 111
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 111
tcagttggca atctcacacc 20
<210> 112
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 112
cctgccactc tgaggaaatg 20
<210> 113
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 113
gcagagtcag tttttcccga 20
<210> 114
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 114
cttaaagctt tctggaagtg 20
<210> 115
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 115
tggaagtcca aaaacctgaa 20
<210> 116
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 116
gagctgcgag gtctggcagc 20
<210> 117
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 117
tacagacata agcaggacct 20
<210> 118
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 118
gaagattgaa ttcctgaagg 20
<210> 119
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 119
attatcctgg aactgatgga 20
<210> 120
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 120
aaggctgtgt ctacttggaa 20
<210> 121
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 121
aaggcctgct cccagttcgg 20
<210> 122
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 122
ttaaactatg tgcaaacagg 20
<210> 123
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 123
agagatgaag caaacaacag 20
<210> 124
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 124
atggaaacga agaaccgaga 20
<210> 125
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 125
aggccttcag gcttgccaga 20
<210> 126
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 126
attctggatg ggtgtttccg 20
<210> 127
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 127
tgctgctgca gtcctgacat 20
<210> 128
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 128
tactgctgac atacagtcgg 20
<210> 129
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 129
tcgaatgcaa tggatgatct 20
<210> 130
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 130
aggcatggac atacttaatg 20
<210> 131
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 131
tctgtgagaa tacactccag 20
<210> 132
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 132
gccagattct gccgaaccaa 20
<210> 133
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 133
cagcgcgttg acttattcat 20
<210> 134
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 134
cacttctgga gacactggat 20
<210> 135
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 135
cgacaaatgt gtgcgatcgg 20
<210> 136
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 136
gctgggactt tggatttcgg 20
<210> 137
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 137
tactgtattg tgttgtcccg 20
<210> 138
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 138
ctggaaatta cctaaacagt 20
<210> 139
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 139
tggatgaatt tcatagacaa 20
<210> 140
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 140
ttcagttagt gtcccgagaa 20
<210> 141
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 141
aaaaggcttt ggttttcagg 20
<210> 142
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 142
aaaggtgaag tgttaaaagt 20
<210> 143
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 143
gtggctgaaa aagagaaagc 20
<210> 144
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 144
tcagaggata ctgcacttgt 20
<210> 145
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 145
gtgcatttca atgaagtcat 20
<210> 146
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 146
gtttcccaat ttctgaccga 20
<210> 147
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 147
tttggtcttc aagtagccaa 20
<210> 148
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 148
gtgcaaagct gccagtgaag 20
<210> 149
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 149
gtttacgtca ggataaggtg 20
<210> 150
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 150
ccttttctga actggtgtcc 20
<210> 151
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 151
tctacggcac gtaccgcaca 20
<210> 152
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 152
aagctcagtg tccgcaaccg 20
<210> 153
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 153
ggggtcggtt ctcccgaatg 20
<210> 154
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 154
cccggtgacc gtgtacgacg 20
<210> 155
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 155
gggcgaccgt acatgactat 20
<210> 156
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 156
cttgctccac ttcaacgtgt 20
<210> 157
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 157
tgccaactgc agcacgctag 20
<210> 158
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 158
ccagctgctt gtaacagtgg 20
<210> 159
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 159
caacatcgca gtggccgtcg 20
<210> 160
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 160
cggcacagct cgtcgcacag 20
<210> 161
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 161
cagctgagat gaccttccgg 20
<210> 162
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 162
aggcagagca gggtacacca 20
<210> 163
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 163
tccgcgagag ccgcaaagtg 20
<210> 164
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 164
acatcctggt agctgagggg 20
<210> 165
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 165
tgtgggagat cgtgacccta 20
<210> 166
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 166
cagatggagt ggacgccgca 20
<210> 167
<211> 20
<212> DNA
<213> Artifical Sequence
<400> 167
gtgccatcag ctctcgtgag 20
<210> 168
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 168
aucauuuucu cagccuccag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 169
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 169
ccucauugcc cucaacacag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 170
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 170
aaauccugca uggcgccgug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 171
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 171
cugucccaau gggagcugcu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 172
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 172
ccgggcaggu cuugacgcag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 173
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 173
ccagauggau gugaaccccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 174
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 174
guguguaacg gaauagguau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 175
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 175
ccaggcaguc gcucucccgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 176
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 176
guggagaucg ccacugaugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 177
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 177
aguuccugug gauccagagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 178
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 178
caugccuugu gcucccccga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 179
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 179
cuugagggag cguaauccca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 180
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 180
ucguucggaa gcgcacgcug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 181
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 181
gcguucggca cgguguauaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 182
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 182
cugcgugaug agcugcacgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 183
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 183
ucgcuuggug caccgcgacc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 184
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 184
cuggcaggga uuccgucaua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 185
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 185
cugaaugaca agguagcgcu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 186
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 186
aaccagcaac aauuccaccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 187
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 187
agaaguccug cugguaguca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 188
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 188
cugcugggcg cgggguucag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 189
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 189
ggacaguguu gagauacucg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 190
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 190
cugaauuagc uguaucguca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 191
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 191
ggaccaguac augaggacug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 192
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 192
gauguaccua ugguccuagu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 193
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 193
aaagacaaga cagagagugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 194
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 194
gggccaggcu cuguucaacg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 195
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 195
ccuauuggac aaauuuggug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 196
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 196
aacaguuauu ggaaucucug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 197
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 197
ggcaggaaga cucuaacgau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 198
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 198
gagagaagaa accaauuggu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 199
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 199
uauaaccaca uguuugacag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 200
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 200
gggcccaaua gaguccgagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 201
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 201
gcgcaucagc ugguucaaag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 202
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 202
guguucagag gaucaaccac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 203
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 203
augauuggga gauuccugau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 204
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 204
uugaaggcuu guaacugcug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 205
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 205
aacuggcuau uguuacccag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 206
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 206
acagugaaau cucgauggag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 207
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 207
cuguuguuga uguuugaaua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 208
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 208
gcgagaauuu ggggaaagag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 209
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 209
ucgggcuggu uuccaaacag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 210
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 210
uuugugauug aauaaggaga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 211
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 211
aaguauaugu gaggcacaug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 212
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 212
caggcaggag ugcauaucuu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 213
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 213
gaggacaaga agaucauggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 214
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 214
ucucaaucuu uagacacugu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 215
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 215
uagaagggaa uaaaaagguu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 216
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 216
acaguuuuuu acagauggaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 217
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 217
guuaagaaua gaaucauucu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 218
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 218
ccaggaagcu uggacgaugu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 219
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 219
uacuaccugu aagccaaucg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 220
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 220
gaguagcuga guggcucccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 221
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 221
guaggucccg ggcguagaca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 222
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 222
gggcgaggag gcgaucuugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 223
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 223
gguggauucg ccaaggcgaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 224
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 224
cgggacuggu cauagcuccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 225
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 225
cuggcgccgc auccccuccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 226
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 226
agcacaccau ccugaguccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 227
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 227
gcccuggaau acaucuccag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 228
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 228
augugccgga aacugccugu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 229
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 229
auggcagguc aggacccuaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 230
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 230
caggcucaag ccuucauagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 231
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 231
ucacauggag agcucuugau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 232
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 232
aaacaaggag cugaaacccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 233
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 233
gggagcgagg gcccccugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 234
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 234
gcccagcaca gacacgccgu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 235
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 235
ccccugauca uugcagccgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 236
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 236
ggggcaggaa uguccuccgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 237
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 237
ggaggcggag gauauauagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 238
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 238
cugcuucugu gaccacggga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 239
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 239
gcagcuccug gugcuuccgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 240
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 240
augaggguga uguuuuuccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 241
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 241
ugaugaucag ggcuuccaug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 242
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 242
ggcgagggcg ggucucucgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 243
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 243
cagggcgagc uacuauagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 244
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 244
uuauccggua uacaggccca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 245
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 245
ucaacaggca aaacgggagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 246
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 246
agcuugugga acccaacgua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 247
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 247
ugcaacuggg agacuuccgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 248
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 248
ccucguaaug accagcucca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 249
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 249
aaagugucgg gagucgugug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 250
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 250
uguguccaga ccguccuaug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 251
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 251
gagcucaagu cccgacacug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 252
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 252
gcaguaaaug aaguugguga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 253
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 253
gguauauagc auccaaccga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 254
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 254
gaccugagaa uuuuuuacag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 255
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 255
ugaggugcac uaauagaggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 256
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 256
cagcaaaggg gaugcgaggu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 257
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 257
gcaccgaaag cuccugcggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 258
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 258
cuggcagaac uggaccuaug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 259
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 259
accaggguag uacccacuga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 260
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 260
uguguggcug cugaauggga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 261
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 261
gcuauuggug aaugacaugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 262
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 262
cuggaccaca cucuauucag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 263
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 263
aaugcacuga uguacuauag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 264
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 264
ggguugcaga auucggugca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 265
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 265
ggucccccug cgguagacug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 266
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 266
gucacuccuu auaccuacug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 267
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 267
aaguuguggu ggaauuuagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 268
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 268
uguaaaggcc cuaaccugga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 269
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 269
ccaagaagag uuaugaggug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 270
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 270
gaugggauau uaugcugaag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 271
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 271
gagaggugaa gauucacaau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 272
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 272
agaaucugga auaaccuuag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 273
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 273
aucugggcaa uggaucugga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 274
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 274
auacuggauc aucacagcaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 275
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 275
agaaacgcuu uaucuggaaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 276
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 276
uggugaugcc auaccaugug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 277
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 277
ccugguggua aauguuccaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 278
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 278
ucaguuggca aucucacacc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 279
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 279
ccugccacuc ugaggaaaug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 280
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 280
gcagagucag uuuuucccga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 281
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 281
cuuaaagcuu ucuggaagug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 282
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 282
uggaagucca aaaaccugaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 283
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 283
gagcugcgag gucuggcagc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 284
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 284
uacagacaua agcaggaccu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 285
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 285
gaagauugaa uuccugaagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 286
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 286
auuauccugg aacugaugga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 287
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 287
aaggcugugu cuacuuggaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 288
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 288
aaggccugcu cccaguucgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 289
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 289
uuaaacuaug ugcaaacagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 290
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 290
agagaugaag caaacaacag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 291
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 291
auggaaacga agaaccgaga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 292
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 292
aggccuucag gcuugccaga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 293
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 293
auucuggaug gguguuuccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 294
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 294
ugcugcugca guccugacau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 295
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 295
uacugcugac auacagucgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 296
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 296
ucgaaugcaa uggaugaucu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 297
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 297
aggcauggac auacuuaaug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 298
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 298
ucugugagaa uacacuccag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 299
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 299
gccagauucu gccgaaccaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 300
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 300
cagcgcguug acuuauucau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 301
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 301
cacuucugga gacacuggau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 302
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 302
cgacaaaugu gugcgaucgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 303
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 303
gcugggacuu uggauuucgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 304
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 304
uacuguauug uguugucccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 305
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 305
cuggaaauua ccuaaacagu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 306
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 306
uggaugaauu ucauagacaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 307
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 307
uucaguuagu gucccgagaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 308
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 308
aaaaggcuuu gguuuucagg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 309
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 309
aaaggugaag uguuaaaagu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 310
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 310
guggcugaaa aagagaaagc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 311
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 311
ucagaggaua cugcacuugu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 312
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 312
gugcauuuca augaagucau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 313
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 313
guuucccaau uucugaccga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 314
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 314
uuuggucuuc aaguagccaa guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 315
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 315
gugcaaagcu gccagugaag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 316
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 316
guuuacguca ggauaaggug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 317
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 317
ccuuuucuga acuggugucc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 318
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 318
ucuacggcac guaccgcaca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 319
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 319
aagcucagug uccgcaaccg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 320
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 320
ggggucgguu cucccgaaug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 321
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 321
cccggugacc guguacgacg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 322
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 322
gggcgaccgu acaugacuau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 323
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 323
cuugcuccac uucaacgugu guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 324
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 324
ugccaacugc agcacgcuag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 325
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 325
ccagcugcuu guaacagugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 326
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 326
caacaucgca guggccgucg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 327
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 327
cggcacagcu cgucgcacag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 328
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 328
cagcugagau gaccuuccgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 329
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 329
aggcagagca ggguacacca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 330
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 330
uccgcgagag ccgcaaagug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 331
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 331
acauccuggu agcugagggg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 332
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 332
ugugggagau cgugacccua guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 333
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 333
cagauggagu ggacgccgca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 334
<211> 100
<212> RNA
<213> Artifical Sequence
<400> 334
gugccaucag cucucgugag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
Claims (10)
1.一种基于CRISPR技术的靶基因捕获的方法，其特征在于其步骤包括：
(1)设计并制备靶基因的sgRNA库，将制备好的sgRNA与Cas蛋白结合，获得sgRNA/Cas复合体；
(2)将sgRNA/Cas复合体与待测样本混合孵育，sgRNA/Cas复合体结合到靶序列的对应位置，获得sgRNA/Cas/靶序列三元复合物；
(3)在被处理样本中加入核酸水解酶，水解非靶基因的序列，然后终止核酸水解酶反应，纯化获得靶基因样本。
2.根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其特征在于：所述Cas蛋白包括但不限于Cas9、Cas12、Cas13、Cas14。
3.根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其特征在于：所述sgRNA库根据靶基因两端的序列进行设计，靶基因序列两端sgRNA间隔不超过10KB。
4.根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其特征在于：步骤(1)中的结合条件为在1×Cas蛋白反应液中以20～40℃条件下结合。
5.根据权利要求4所述的基于CRISPR技术的靶基因捕获的方法，其特征在于，所述的Cas蛋白反应液中含有Tris-HCl、NaCl、MgCl2、BSA和Tween；
所述的Tris-HCl浓度为10mM～200mM；
所述的NaCl浓度为10mM～200mM；
所述的MgCl2浓度为1mM～50mM；
所述的BSA浓度为10ng/μL～50ng/μL；
所述的Tween浓度为0.01％～2％体积比。
6.根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其特征在于：步骤(2)中孵育温度为25℃～50℃。
7.根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其特征在于：步骤(3)中的核酸水解酶为大肠杆菌外切酶1、大肠杆菌外切酶3、大肠杆菌外切酶7、噬菌体核酸外切酶、T7噬菌体基因Ⅵ核酸外切酶或Bal 31核酸酶。
8.根据权利要求7所述的基于CRISPR技术的靶基因捕获的方法，其特征在于，所述的核酸水解酶使用量为1U/rxn～1000U/rxn。
9.根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其特征在于，步骤(3)中在25℃～50℃条件下进行水解。
10.根据权利要求1所述的基于CRISPR技术的靶基因捕获的方法，其特征在于：所述sgRNA库的制备通过以下步骤制得：
A、合成T7启动子-gRNA-Cas蛋白骨架的DNA序列；
B、使用T7启动子-gRNA-Cas蛋白骨架DNA序列的5端重叠区域为上游引物，通用Cas蛋白骨架DNA序列的3端序列作为下游引物，添加Cas蛋白骨架DNA作为模板，扩增得到T7启动子-gRNA-Cas蛋白骨架DNA；
C、使用T7体外转录获得sgRNA库。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210367292.7A CN114836411A (zh) | 2022-04-08 | 2022-04-08 | 基于crispr技术的靶基因捕获的方法 |
| PCT/CN2023/086511 WO2023193748A1 (zh) | 2022-04-08 | 2023-04-06 | 基于crispr技术的靶基因捕获的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210367292.7A CN114836411A (zh) | 2022-04-08 | 2022-04-08 | 基于crispr技术的靶基因捕获的方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114836411A true CN114836411A (zh) | 2022-08-02 |
Family
ID=82563559
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210367292.7A Pending CN114836411A (zh) | 2022-04-08 | 2022-04-08 | 基于crispr技术的靶基因捕获的方法 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114836411A (zh) |
| WO (1) | WO2023193748A1 (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023193748A1 (zh) * | 2022-04-08 | 2023-10-12 | 翌圣生物科技(上海)股份有限公司 | 基于crispr技术的靶基因捕获的方法 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355289A (zh) * | 2018-12-24 | 2019-02-19 | 人和未来生物科技（长沙）有限公司 | 用于从预文库中富集靶标序列的试剂盒、制备方法和应用 |
| US20200181683A1 (en) * | 2017-08-08 | 2020-06-11 | Depixus | In vitro isolation and enrichment of nucleic acids using site-specific nucleases |
| WO2020120711A1 (en) * | 2018-12-12 | 2020-06-18 | Depixus | Method of nucleic acid enrichment using site-specific nucleases followed by capture |
| CN113330122A (zh) * | 2018-11-16 | 2021-08-31 | 德皮克斯公司 | 使用位点特异性核酸酶优化核酸的体外分离 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114836411A (zh) * | 2022-04-08 | 2022-08-02 | 翌圣生物科技(上海)股份有限公司 | 基于crispr技术的靶基因捕获的方法 |
-
2022
- 2022-04-08 CN CN202210367292.7A patent/CN114836411A/zh active Pending
-
2023
- 2023-04-06 WO PCT/CN2023/086511 patent/WO2023193748A1/zh unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200181683A1 (en) * | 2017-08-08 | 2020-06-11 | Depixus | In vitro isolation and enrichment of nucleic acids using site-specific nucleases |
| CN113330122A (zh) * | 2018-11-16 | 2021-08-31 | 德皮克斯公司 | 使用位点特异性核酸酶优化核酸的体外分离 |
| US20210388414A1 (en) * | 2018-11-16 | 2021-12-16 | Depixus | Optimization of in vitro isolation of nucleic acids using site-specific nucleases |
| WO2020120711A1 (en) * | 2018-12-12 | 2020-06-18 | Depixus | Method of nucleic acid enrichment using site-specific nucleases followed by capture |
| CN109355289A (zh) * | 2018-12-24 | 2019-02-19 | 人和未来生物科技（长沙）有限公司 | 用于从预文库中富集靶标序列的试剂盒、制备方法和应用 |
Non-Patent Citations (3)
| Title |
|---|
| STEVENS RC 等: "\"A novel CRISPR/Cas9 associated technology for sequence-specific nucleic acid enrichment\"", 《PLOS ONE》 * |
| ZHUANG XW 等: "\"Review-CRISPR/Cas Systems: Endless Possibilities for Electrochemical Nucleic Acid Sensors\"", 《JOURNAL OF THE ELECTROCHEMICAL SOCIETY》 * |
| 杨鹏: "\"运用CRISPR-dCas9技术在原代培养T细胞中诱导Foxp3基因过表达和调节性T细胞分化\"", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023193748A1 (zh) * | 2022-04-08 | 2023-10-12 | 翌圣生物科技(上海)股份有限公司 | 基于crispr技术的靶基因捕获的方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023193748A1 (zh) | 2023-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110699426B (zh) | 基因目标区域富集方法及试剂盒 | |
| CN109593757B (zh) | 一种探针及其适用于高通量测序的对目标区域进行富集的方法 | |
| CN110878298B (zh) | rRNA捕获探针及其应用 | |
| CN113774032B (zh) | 重组t4连接酶突变体、编码dna及ngs建库方法 | |
| CN110607353B (zh) | 一种利用高效地连接技术快速制备dna测序文库的方法和试剂盒 | |
| CN112176031A (zh) | 一种去核糖体rna测序文库的构建方法及试剂盒 | |
| CN112251821A (zh) | 一种快速高效的构建二代测序文库的试剂盒 | |
| CN107904667A (zh) | 一种新型甲基化建库试剂盒及其应用 | |
| CN110699425B (zh) | 基因目标区域的富集方法及体系 | |
| CN108166068A (zh) | 一种新型dna建库试剂盒及其应用 | |
| CN114836411A (zh) | 基于crispr技术的靶基因捕获的方法 | |
| CN113943763B (zh) | 一种还原核酸的方法及其检测方法 | |
| JPS63500006A (ja) | エキソヌクレア−ゼ阻害による核酸の塩基配列決定法 | |
| CN116043337A (zh) | Dna甲基化标志物筛查试剂盒及方法 | |
| CN113699214A (zh) | 基于基因捕获技术的测序方法 | |
| CN112941635A (zh) | 一种提高文库转化率的二代测序建库试剂盒及其方法 | |
| CN108166069A (zh) | 一种新型甲基化建库方法及其应用 | |
| CN108166067A (zh) | 一种新型dna建库方法及其应用 | |
| CN111989406A (zh) | 一种测序文库的构建方法 | |
| CN112501249A (zh) | Rna文库的制备方法、测序方法和试剂盒 | |
| CN113817803B (zh) | 一种携带修饰的小rna的建库方法及其应用 | |
| CN113862333B (zh) | 一种组合物及其氧化5-甲基胞嘧啶的方法 | |
| CN114807084A (zh) | 突变型Tn5转座酶及试剂盒 | |
| CN109852668A (zh) | 一种简化基因组测序文库及其建库方法 | |
| CN112458085A (zh) | 一种新型分子捕获优化探针及其文库构建方法 |
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 |