CN112852884B - 一种环形rna敲低载体快速构建试剂盒及其应用 - Google Patents
一种环形rna敲低载体快速构建试剂盒及其应用 Download PDFInfo
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Abstract
本发明属于分子生物学技术领域，具体涉及一种环形RNA敲低载体快速构建试剂盒及其应用。本发明提供的试剂盒，通过调节试剂组分、配方和连接体系中空载体与gRNA的比例等，最终制成的试剂盒中共包含Linearized CasRx Vector、DNA Ligase Mix、10×Ligase Buffer、Nuclease‑free Water、U6Primer(10μM)、gRNA‑NC Vector等6种成分，采用本发明提供的试剂盒，可以快速高效构建含CasRx蛋白和gRNA表达的整合型载体，解决了现有技术中使用CRISPR/CasRx系统敲低环形RNA时碰到的质粒共转染效率低、敲低效果有限等问题。
Description
技术领域
本发明属于分子生物学技术领域，具体涉及一种环形RNA敲低载体快速构建试剂盒及其应用。
背景技术
环形RNA(circular RNAs,circRNAs)是一类具有闭合环状结构的RNA分子，在多物种中广泛存在，比线性RNA更稳定。由于环形RNA与亲本基因来源于同一pre-mRNA，具有相同的外显子序列，只在反向剪切位点有特异序列，使得环形RNA敲除和敲低的实验设计存在难度高、受限多等问题。
目前对环形RNA敲低研究中广泛应用的是siRNA/shRNA干扰，但环形RNA与亲本RNA有相同的序列，仅在环化位点处序列有特异性，很容易非特异靶向亲本RNA或其他RNA。siRNA靶标设计难度也很高，只能限定长度19～21nt且靶向反向剪切位点，对序列要求高，设计受限，可选择性低，部分序列特殊的环形RNA甚至无法设计出一条siRNA。因此使用siRNA/shRNA敲低环形RNA经常碰到干扰效率低、脱靶和潜在脱靶或错误靶向亲本基因mRNA等结果，直接影响实验结果的可靠性。
另外也有使用ASO(反义寡核苷酸)干扰circRNA的，存在的问题是ASO主要在细胞核内发挥作用，难以适用于大部分定位于细胞质的circRNA。而针对反向剪切位点设计靶标的限制与siRNA基本相同。
CRISPR-Cas13系统是以Cas13酶作为Cas核酸酶的CRISPR子系统，仅靶向RNA而不靶向DNA，具有靶向效率高、酶切能力优异的特点。目前已经发现四类Cas13蛋白，分别为Cas13a、Cas13b、Cas13c和Cas13d，具有RNA酶活性，精确切割RNA，而且切割位点具有序列依赖性和结构依赖性。
研究(Screening for functional circular RNAs using the CRISPR–Cas13system.https://doi.org/10.1038/s41592-020-01011-4.)利用CRISPR/RfxCas13d(CasRx)系统成功地批量化敲低了环形RNA，为环形RNA敲低验证了一种新的技术选择。但该研究中CRISPR/CasRx(RfxCas13d)系统是设计为CasRx(RfxCas13d)蛋白表达载体和gRNA表达载体两个质粒共同使用的，且CasRx(RfxCas13d)蛋白表达载体的序列构建为RfxCas13d-mCherry-Flag，增加了mCherry蛋白与CasRx融合表达以保持CasRx的稳定，因此CasRx蛋白表达载体构建步骤繁琐且序列较长，导致质粒长度过大。使用时需要将CasRx蛋白表达载体和gRNA表达载体两个质粒共转染，因为涉及两个质粒共转染，且质粒较大，因此很容易导致转染效率偏低，表达量有限，从而影响敲低实验效果。此外利用CasRx敲低其他RNA的研究中，也是CasRx蛋白表达载体和gRNA表达载体两个质粒共同使用，同样容易碰到转染效率低和敲低实验效果有限的问题。
因此需要开发一种能快速构建CRISPR/CasRx整合型载体的试剂盒用于环形RNA敲低研究。
发明内容
针对现有技术普遍存在的缺陷，本发明创造性地提供了一种环形RNA敲低载体快速构建试剂盒及其应用。采用本发明提供的试剂盒可以快速高效构建含CasRx蛋白和gRNA表达的整合型载体，解决了现有技术中使用CRISPR/CasRx系统敲低环形RNA时碰到的质粒共转染效率低、敲低效果有限等问题。
为了达到上述目的，本发明采用的技术方案为：
一种环形RNA敲低载体快速构建的试剂盒，包含Linearized CasRx Vector、DNALigase Mix、10×Ligase Buffer、Nuclease-free Water、10μM的U6 Primer、gRNA-NCVector共6种成分。本发明提供的试剂盒的试验流程图如图1所示。
优选地，所述Linearized CasRx Vector选自Linearized CasRx Vector，FrompCasRx-L1、Linearized CasRx Vector，From pCasRxN-L1、Linearized CasRx Vector，From pCasRx-A1和Linearized CasRx Vector，From pCasRxN-A1中的一个或多个。
优选地，所述Linearized CasRx Vector的序列组成为TTTT-U6terminator-CMVpromoter-CasRx-Flag-EGFP-IRES-PuroR-WPRE-backbone-U6promoter-CasRx DR30-AAAC；所述Linearized CasRx Vector，From pCasRx-L1和Linearized CasRx Vector，FrompCasRxN-L1为慢病毒载体骨架；所述Linearized CasRx Vector，From pCasRx-A1和Linearized CasRx Vector，From pCasRxN-A1为ssAAV载体骨架；所述Linearized CasRxVector，From pCasRxN-L1和Linearized CasRx Vector，From pCasRxN-A1中CasRx的5’和3’末端分别添加有核定位信号NLS序列；所述Linearized CasRx Vector，From pCasRx-L1的核苷酸序列如SEQ ID NO.1；所述Linearized CasRx Vector，From pCasRxN-L1的核苷酸序列如SEQ ID NO.2；所述Linearized CasRx Vector，From pCasRx-A1的核苷酸序列如SEQID NO.3；所述Linearized CasRx Vector，From pCasRxN-A1的核苷酸序列如SEQ ID NO.4。
优选地，所述Linearized CasRx Vector，From pCasRx-L1为pCasRx-L0经BpiI双酶切线性化后回收制备而成；所述Linearized CasRx Vector，From pCasRxN-L1为pCasRxN-L0经BpiI双酶切线性化后回收制备而成；所述Linearized CasRx Vector，FrompCasRx-A1为pCasRx-A0经BpiI双酶切线性化后回收制备而成；所述Linearized CasRxVector，From pCasRxN-A1为pCasRxN-A0经BpiI双酶切线性化后回收制备而成。
优选地，所述DNA Ligase Mix包含500U/μL的T4 DNA ligase、pH为7.5，20mM的Tris-HCl、50mM KCl、1mM DTT、0.1mM EDTA和50％甘油；所述10×Ligase Buffer包含pH为7.8，400mM Tris-HCl、100mM MgCl2、100mM DTT、5mM ATP和25％PEG 4000；所述10μM的U6Primer为合成的U6引物，核苷酸序列信息如SEQ ID NO.5所示，并经PAGE纯化，使用时Nuclease-free Water稀释。
ATGGACTATCATATGCTTACCGTA(SEQ ID NO.5)；
优选地，所述gRNA-NC Vector选自gRNA-NC Vector,pCasRx-L1、gRNA-NC Vector,pCasRxN-L1、gRNA-NC Vector,pCasRx-A1和gRNA-NC Vector,pCasRxN-A1中的一个或多个；4种载体的区别及对应的试剂盒货号如表1所示，载体图谱如图2所示。
表1 4种载体的区别及对应的试剂盒货号
优选地，所述gRNA-NC Vector的序列组成为backbone-U6 promoter-CasRx DR30-non-targeting gRNA-U6 terminator-CMV promoter-CasRx-Flag-EGFP-IRES-PuroR-WPRE；所述gRNA-NC Vector，pCasRx-L1和gRNA-NC Vector，pCasRxN-L1为慢病毒载体骨架；所述gRNA-NC Vector，pCasRx-A1和gRNA-NC Vector，pCasRxN-A1为ssAAV载体骨架；所述gRNA-NC Vector，pCasRxN-L1和gRNA-NC Vector，pCasRxN-A1中CasRx的5’和3’末端分别添加有核定位信号NLS序列；所述gRNA-NC Vector，pCasRx-L1的核苷酸序列如SEQ ID NO.6；所述gRNA-NC Vector，pCasRxN-L1的核苷酸序列如SEQ ID NO.7；所述gRNA-NC Vector，pCasRx-A1的核苷酸序列如SEQ ID NO.8；所述gRNA-NC Vector，pCasRxN-A1的核苷酸序列如SEQ ID NO.9。
本发明还提供了一种利用所述试剂盒在构建目标环形RNA的CasRx/gRNA整合型表达载体中的应用。
本发明还提供了一种CasRx/gRNA整合型表达载体在制备可持续敲低目标环形RNA的稳定细胞系、动物体或组织脏器模型中的应用，所述CasRx/gRNA整合型表达载体采用所述试剂盒制成。
本发明提供的试剂盒，是经过反复测试和验证，并调节试剂组分、配方和连接体系中空载体与gRNA的比例，获得基于CasRx/gRNA整合型载体的快速构建试剂盒，提供整合型通用NC载体，以及构建目标gRNA载体的连接酶、buffer和线性化载体等试剂，配套齐全。
使用本发明中试剂盒构建的目标环形RNA的CasRx/gRNA表达载体，可以瞬时转染细胞后以RT-qPCR检测环形RNA的表达丰度，用于快速批量验证高效的环形RNA敲低gRNA设计；还可以包装慢病毒后感染细胞，获得持续敲低目标环形RNA的稳定细胞系，用于目标环形RNA的功能和机制研究；还可以包装ssAAV病毒后感染动物体，获得持续敲低目标环形RNA的动物体或组织脏器模型，用于环形RNA的体内功能和机制研究。
与现有技术相比，本发明提供的环形RNA敲低载体快速构建的试剂盒具有如下优势：
(1)本发明提供的试剂盒，使用时无需酶切，通过一步反应，在30分钟内即可快速完成CasRx/gRNA载体连接；
(2)本发明提供的试剂盒，用户只需要合成两条gRNA引物序列用于连接即可，成本低，操作简便；
(3)本发明提供的试剂盒，构建效率高，限定优化的反应体系保证阳性率高，大幅提高载体构建成功率。
附图说明
图1为本发明试剂盒实验流程图；
图2为4种载体空载体图谱；
图3为gRNA-F和gRNA-R退火形成的双链产物示意图；
图4为载体中表达crRNA的序列示意图；
图5为三个gRNA载体转化平板菌落生长图；
图6a、6b及6c为30个单克隆菌液测序峰图对比图；
图7为hsa_circ_0001821敲低后的表达丰度图；
具体实施方式
下面结合具体实施例对本发明作进一步解释，但是应当注意的是，以下实施例仅用以解释本发明，而不能用来限制本发明，所有与本发明相同或相近的技术方案均在本发明的保护范围之内。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料为市售商品。
术语“环形RNA”、“环状RNA”与“circRNA”可互换使用；本文中，除非特别注明，其他实验方法都可采用本领域中的常规方法，例如参考《分子克隆实验指南(第四版)》(科学出版社，2017)或按照供应商所建议的条件。
实施例1
以Linearized CasRx Vector,From pCasRx-L1为例，示意试剂Linearized CasRxVector，From pCasRxN-L1、Linearized CasRx Vector，From pCasRx-A1和LinearizedCasRx Vector，From pCasRxN-A1的制备方法。
过渡载体13d-GW1载体构建：
以全基因合成法合成GW1-seq序列，作为PCR的模板，GW1的序列信息如SEQIDNO.10所示：
合成如下引物，使用ddH2O溶解为浓度10μM，分别扩增13d-GW1-D1、13d-GW1-D2和13d-GW1-D3。
先扩增207bp，标记为13d-GW1-D1。
13d-GW1-UnF:AGTGAACGGATCTCGACGGTTAACTT(SEQ ID NO.11)；
13d-GW1-D1-R:GTAGATGTACTGCCAAGTAGGA(SEQ ID NO.12)；
再以巢式PCR扩增326bp，标记为13d-GW1-D2。其中以13d-GW1-D2-F和13d-GW1-D2-inR进行巢式PCR的第一轮扩增，以13d-GW1-D2-F和13d-GW1-D2-R进行巢式PCR的第二轮扩增。
13d-GW1-D2-F：CCTACTTGGCAGTACATCTACGCTAGCGAGGGCCTATTTCCCATGATTCC(SEQID NO.13)；
13d-GW1-D2-inR：CAAACCCCGACCAGTTGGTAGGGGTTCGGTGTTTCGTCCTTTCCACAA(SEQID NO.14)；
13d-GW1-D2-R：AAGGTCTTCTCGAAGACCCGTTTCAAACCCCGACCAGTTGGTAGGGGT(SEQ IDNO.15)；
最后扩增357bp，标记为13d-GW1-D3。
13d-GW1-D3-F：GAAACGGGTCTTCGAGAAGACCTTTTTTTTGAATTCGTATTAGTCATCGCTATTACC(SEQ ID NO.16)；
13d-GW1-UnR：GATTTAAATTCGAATTCGCTAGCTCTAGAATC(SEQ ID NO.17)；
等摩尔比混合三个PCR产物，使用13d-GW1-UnF和13d-GW1-UnR扩增全长840bp，PCR产物标记为13d-GW1-D123。
PCR扩增反应程序：95℃5min，38个循环(98℃10s，58℃30s，68℃60s)，68℃5min，4℃保存。反应体系如表2所示；其中的Primer-F及Primer-R分别为上述不同的引物序列。
表2 PCR扩增反应体系
| 试剂 | 体积 |
| 10×PCR Buffer for KOD-Plus-Neo | 5μL |
| KOD-Plus-Neo(1U/μL) | 1μL |
| MgSO<sub>4</sub>(25mM) | 3μL |
| dNTP(2mM) | 5μL |
| Primer-F(10μM) | 1.5μL |
| Primer-R(10μM) | 1.5μL |
| 13d-GW1-D1+13d-GW1-D2+13d-GW1-D3 | 0.4+0.64+0.7μL |
| ddH<sub>2</sub>O | Up to 50μL |
使用FD KspAI/XbaI双酶切PCDH-CMV-MCS-EF1a-GFP-puro(Clontech,CD513B-1)载体，得到开环空载体。
连接PCR产物13d-GW1-D123和开环空载体PCDH-CMV-MCS-EF1a-GFP-puro(慢病毒载体backbone)，构建载体经sanger测序鉴定正确，标记为13d-GW1。
参考
HD Cloning Kit(TaKaRa)说明书配置连接反应液，50℃温育15min，然后进行转化或于-20℃保存，反应条件如表3所示。
表3连接反应体系
| 试剂 | 体积 |
| 13d-GW1-D123 | 1μL |
| PCDH-CMV-MCS-EF1a-GFP-puro开环空载体 | 2μL |
| 5x In-Fusion HD Enzyme Premix | 2μL |
| ddH<sub>2</sub>O | up to 10μL |
即使用慢病毒载体backbone，引入U6 promoter-CasRx DR30-2x BpiI spacer-U6terminator的序列。
过渡载体13d-GW2载体构建：
以全基因合成法合成GW2-seq序列，作为PCR的模板；所述GW2-seq序列信息如SEQID NO.18所示。
使用如下引物分别扩增13d-GW2-D1、13d-GW2-D2和13d-GW2-D3。
先扩增605bp，标记为13d-GW2-D1
13d-GW2-UnF：AGCGAATTCGAATTTAAATCGGATCCTTCGAAGCCCCTCTCCCTCCCCCCCCCCTAACG(SEQ ID NO.19)；
13d-GW2-D1-R：TTATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTG(SEQ ID NO.20)；
扩增651bp，标记为13d-GW2-D2。
13d-GW2-D2-F：TTCCTTTGAAAAACACGATGATAAGCTTGCCAATGACCGAGTACAAGCCCACGGT(SEQ ID NO.21)；
13d-GW2-D2-R：GGTTGATTGCGGCCGCTTATCAGGCACCGGGCTTGCGGGTCA(SEQ IDNO.22)；
扩增630bp，标记为13d-GW2-D3。
13d-GW2-D3-F：TGATAAGCGGCCGCAATCAACCTCTGGATTACAAAATTTGT(SEQ ID NO.23)；
13d-GW2-UnR：CTTGTAAGTCATTGGTCTTAAAGGTACCAGG(SEQ ID NO.24)；
等摩尔比混合三个PCR产物，使用13d-GW2-UnF和13d-GW2-UnR扩增全长1886bp，PCR产物标记为13d-GW2-D123。PCR扩增同上述方法。
使用FD BamHI/KpnI双酶切13d-GW1载体，得到开环空载体。
使用
HD Cloning Kit(TaKaRa)试剂盒，同上述方法，连接PCR产物13d-GW2-D123和开环空载体13d-GW1，构建载体经sanger测序鉴定正确，标记为13d-GW2。即使用慢病毒载体backbone，在引入U6 promoter-CasRx DR30-2x BpiI spacer-U6 terminator的基础上，再引入IRES-PuroR-WPRE序列。过渡载体pCasRx-L0(GS0114)载体构建：
以全基因合成法合成13d-CasRx序列，作为PCR的模板，所述13d-CasRx的序列如SEQ ID NO.25所示；
使用如下引物分别扩增13d-CasRx-D1和13d-CasRx-D2。
扩增2960bp，标记为13d-CasRx-D1
13d-GW3-D1-UnF：TGTTTTGACCTCCATAGAAGATTCTAGAGCCACCATGATCGAAAAAAAAAAGTCCTTCG(SEQ ID NO.26)；
13d-GW3-D1-R：ATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCGGATCCGGAATTGCCGGACACCT(SEQ ID NO.27)；
以巢式PCR扩增880bp，标记为13d-CasRx-D2。巢式PCR扩增同上述方法。
13d-GW3-D2-F：AAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGA(SEQ IDNO.28)；
13d-GW3-D2-F-in：GATCATGACATCGATTACAAGGATGACGATGACAAGGGTAGCGGCAGTGGAGAGGGCAG(SEQ ID NO.29)；
13d-GW3-D2-UnR：TAACGTTAGGGGGGGGGGAGGGAGAGGGGCTTCGAATTACTTGTACAGCTCGTCCATGC(SEQ ID NO.30)；
等摩尔比混合两个PCR产物，使用13d-GW3-D1-UnF和13d-GW3-D2-UnR扩增全长3840bp，PCR产物标记为13d-CasRx-D12。PCR扩增同上述方法。
使用FD XbaI/BamHI双酶切13d-GW2载体，得到开环空载体。
使用
HD Cloning Kit(TaKaRa)试剂盒，同上述方法，连接PCR产物13d-CasRx-D12和开环空载体13d-GW2，构建载体经sanger测序鉴定正确，标记为pCasRx-L0(GS0114)。即使用慢病毒载体backbone，在引入U6promoter-CasRx DR30-2x BpiI spacer-U6 terminator和IRES-PuroR-WPRE序列的基础上，再引入CMV promoter-(NLS)CasRx(NLS)-Flag-(EGFP)序列。
试剂Linearized CasRx Vector,From pCasRx-L1的制备：
向pCasRx-L0(GS0114)载体中加入内切酶FD BpiI进行双酶切。反应体系如表4所示，保持37℃，2h。
表4双酶切反应体系
| 试剂 | 用量 |
| pCasRx-L0(GS0114) | 5μg |
| FD BpiI | 6μL |
| 10X FastDigest Buffer | 6μL |
| ddH<sub>2</sub>O | up to 60μL |
向酶切反应液中加入0.9倍体积的Agencourt AMPure XP(Beckman)磁珠，涡旋混匀后室温静置5分钟，置于磁力架上静置3分钟；吸弃上清，加入2.2倍体积的80％EtOH，磁力架上静置3分钟；吸弃上清，加入2.2倍体积的80％EtOH，磁力架上静置3分钟；吸弃上清，从磁力架上取下，加入50μL Nuclease-free Water，涡旋混匀后室温静置5分钟；置于磁力架上静置3分钟，吸取上清保存，标记为Linearized CasRx Vector，From pCasRx-L1原液。
使用Qubit 3.0Fluorometer(Thermo)测量Linearized CasRx Vector，FrompCasRx-L1原液的浓度，然后使用Nuclease-free Water稀释为浓度10ng/μL，吸取40μL分装并标记即得试剂盒中试剂Linearized CasRx Vector，From pCasRx-L1。
实施例2
以gRNA-NC Vector，pCasRx-L1为例，示意试剂gRNA-NC Vector,pCasRxN-L1、gRNA-NC Vector,pCasRx-A1和gRNA-NC Vector,pCasRxN-A1的制备方法。
1.gRNA双链引物合成与双链退火
设计合成non-targeting gRNA：TCACCAGAAGCGTACCATACTC(SEQ ID NO.31)的双链引物，添加连接定位序列，然后进行引物合成，选择PAGE纯化。gRNA-NT-F：5’-AAACTCACCAGAAGCGTACCATACTC-3’(SEQ ID NO.32)；gRNA-NT-R：5’-AAAAGAGTATGGTACGCTTCTGGTGA-3’(SEQ ID NO.33)；
将合成的双链引物分别加水稀释至浓度50μM，在PCR管中吸取1μL gRNA-NT-F和1μL gRNA-NT-R，再加入8μL ddH2O，然后设置95℃5min，取出后静置3min以降至室温。
2.连接反应
按下表5配置试剂，使用含热盖的PCR仪进行连接反应，设置22℃25min。
表5连接反应体系
| 试剂 | 体积 |
| 双链产物 | 1μL |
| DNA Ligase Mix | 0.5μL |
| 10×Ligase Buffer | 0.5μL |
| Linearized CasRx Vector，From pCasRx-L1 | 2μL |
| Nuclease-free Water | 1μL |
| Total | 5μL |
将连接产物转化DH5a感受态细胞后，挑选单克隆进行sanger测序，鉴定为序列正确的阳性克隆后提取质粒，吸取2μg分装标记即得试剂盒中试剂gRNA-NC Vector，pCasRx-L1。
实施例3本发明提供的试剂盒
本发明提供一种基于CasRx/gRNA整合型载体的快速构建试剂盒，所述试剂盒包含Linearized CasRx Vector、DNA Ligase Mix、10×Ligase Buffer、Nuclease-free Water、U6 Primer(10μM)、gRNA-NC Vector共六种试剂和使用说明。
依据所述试剂盒中不同的四种质粒，制备四种试剂盒的货号和试剂组分如表6。
表6不同质粒制成的试剂盒成分对比
实验流程主要包含以下步骤：
1.1gRNA设计与合成：在靶标RNA内搜索合适的gRNA target序列，无需限定PFS。对于mRNA或lncRNA，gRNA target基本可设计位于任意位置。对于circRNA，gRNA target应设计靶向反向剪切位点(back-splicing junction,BSJ)处。
设计原则可参考文章(Wessels HH et al.,2020)或Cas13design在线工具(https://cas13design.nygenome.org/#)。设计靶向的RNA序列为gRNA target序列，将其反向互补后得到gRNA序列。
1.2gRNA合成：给gRNA添加连接定位序列后，分别合成gRNA-F和gRNA-R序列，选择PAGE纯化。
gRNA-F：5’-AAAC+gRNA-3’；gRNA-R：5’-AAAA+gRNA(antisense)-3’。
设计与合成实例
1.3设计与合成实例：对如下RNA序列设计，靶向下划线位置，则对应序列分别如下。
>Sample RNA|5’-3’
TTTGCCTTCCTGTTTTTGCTGAGTATGGTACGCTTCTGGTGACACCCAGAAA
CGCTGGTG
>gRNA target|5’-3’
GAGTATGGTACGCTTCTGGTGA
>gRNA|5’-3’
TCACCAGAAGCGTACCATACTC
需要合成的gRNA-F和gRNA-R序列如下。
gRNA-F：5’-AAACTCACCAGAAGCGTACCATACTC-3’
gRNA-R：5’-AAAAGAGTATGGTACGCTTCTGGTGA-3’
2.gRNA双链退火
2.1退火反应：将合成的gRNA-F和gRNA-R分别加水稀释至浓度50μM。按下表7配置试剂，使用含热盖的PCR仪进行退火反应，设置95℃5min，取出后静置3min以降至室温，立即进行连接反应或于-20℃保存待用。
表7退火反应体系
| 试剂 | 体积 |
| gRNA-F(50μM) | 1μL |
| gRNA-R(50μM) | 1μL |
| ddH<sub>2</sub>O | 8μL |
| Total | 10μL |
2.2步骤1.3中合成的gRNA-F和gRNA-R退火形成的双链产物如图3所示。
3.连接
3.1连接反应
按下表8配置试剂，使用含热盖的PCR仪进行连接反应，设置22℃25min，取出后立即进行转化涂板或于-20℃保存待用。
表8连接反应体系
3.2连接反应实例：以步骤2.2中的退火双链产物连接载体，载体中表达crRNA的序列如图4所示。
4.连接产物转化涂板
按照使用的感受态细胞说明书进行转化实验，主要步骤参考如下：
(1)在冰上解冻感受态细胞(DH5α、TOP10或Stbl3等)；
(2)将5μL连接产物加入50μL感受态细胞中，轻弹管壁混匀，在冰上放置30min；
(3)42℃热激45s，热激时避免摇动离心管，取出后迅速置于冰上放置2min；
(4)加入500μL SOC或LB培养基，混匀后以37℃，200rpm培养1h，以使细菌复苏；
(5)吸取适量培养液于LB(Amp+)琼脂培养基上，使用涂布棒轻轻涂匀；
(6)将平板置于37℃至菌液被吸收，然后倒置平板，37℃过夜培养。
5.克隆鉴定：可使用菌落PCR或测序鉴定阳性克隆。菌落PCR鉴定简便快捷，测序可验证序列和位置的准确性。
5.1菌落PCR鉴定
挑取单个菌落至20～50μL LB(Amp+)培养基中混匀，取1～2μL作为PCR的模板。PCR可使用U6 Primer(10μM)和合成的gRNA-R进行扩增，阳性克隆的扩增大小约为135bp，注意按需调节引物浓度。
5.2测序鉴定
使用通用引物U6进行测序鉴定。
实施例4
针对hsa_circ_0001821设计3条gRNA，分别合成gRNA双链，使用C0101-RL试剂盒构建CasRx/gRNA载体，其余实验步骤同实施例3。
hsa_circ_0001821-gRNA1-F:AAACAAAGATCAGGCCTCAAGCCCAG(SEQID NO.34)
hsa_circ_0001821-gRNA1-R:AAAACTGGGCTTGAGGCCTGATCTTT(SEQ ID NO.35)
hsa_circ_0001821-gRNA2-F:AAACCAAAAGATCAGGCCTCAAGCCCAGC(SEQ ID NO.36)
hsa_circ_0001821-gRNA2-R:AAAAGCTGGGCTTGAGGCCTGATCTTTTG(SEQ ID NO.37)
hsa_circ_0001821-gRNA3-F：AAACCCAAAAGATCAGGCCTCAAGCCCAGCTG(SEQ IDNO.38)
hsa_circ_0001821-gRNA3-R：AAAACAGCTGGGCTTGAGGCCTGATCTTTTGG(SEQ IDNO.39)
三个gRNA载体转化平板菌落结果如图5所示，分别从三个转化平板上各挑取10个，共30个单克隆菌液进行sanger测序鉴定，使用通用引物U6，结果显示29个为含正确序列的阳性克隆，阳性率为96.7％。测序峰图对比如图6a、6b及6c，其中深色选中部分为正确的阳性序列。
实施例5
针对hsa_circ_0001821设计构建的三条CasRx/gRNA载体，按照hsa_circ_0001821-gRNA1、hsa_circ_0001821-gRNA2、hsa_circ_0001821-gRNA3、gRNA-NC Vector和293T共5组，使用Lipofectamine 3000转染293T细胞，48h后消化离心收取样品。
细胞样品抽提总RNA后，进行逆转录合成cDNA，然后进行qPCR检测hsa_circ_0001821的表达丰度，以2-△△Ct公式计算。结果如图7，显示hsa_circ_0001821-gRNA1、hsa_circ_0001821-gRNA2和hsa_circ_0001821-gRNA3都能有效敲低hsa_circ_0001821的表达，其中hsa_circ_0001821-gRNA3具有最优的敲低效率。
序列表
<110> 广州吉赛医疗科技有限公司
<120> 一种环形RNA敲低载体快速构建试剂盒及其应用
<130> 2021.1.25
<160> 39
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11447
<212> DNA
<213> Linearized CasRx Vector，From pCasRx-L1
<400> 1
tttttttgaa ttcgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 60
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 120
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 180
cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 240
tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag 300
aagattctag agccaccatg atcgaaaaaa aaaagtcctt cgccaagggc atgggcgtga 360
agtccacact cgtgtccggc tccaaagtgt acatgacaac cttcgccgaa ggcagcgacg 420
ccaggctgga aaagatcgtg gagggcgaca gcatcaggag cgtgaatgag ggcgaggcct 480
tcagcgctga aatggccgat aaaaacgccg gctataagat cggcaacgcc aaattcagcc 540
atcctaaggg ctacgccgtg gtggctaaca accctctgta tacaggaccc gtccagcagg 600
atatgctcgg cctgaaggaa actctggaaa agaggtactt cggcgagagc gctgatggca 660
atgacaatat ttgtatccag gtgatccata acatcctgga cattgaaaaa atcctcgccg 720
aatacattac caacgccgcc tacgccgtca acaatatctc cggcctggat aaggacatta 780
ttggattcgg caagttctcc acagtgtata cctacgacga attcaaagac cccgagcacc 840
atagggccgc tttcaacaat aacgataagc tcatcaacgc catcaaggcc cagtatgacg 900
agttcgacaa cttcctcgat aaccccagac tcggctattt cggccaggcc tttttcagca 960
aggagggcag aaattacatc atcaattacg gcaacgaatg ctatgacatt ctggccctcc 1020
tgagcggact gaggcactgg gtggtccata acaacgaaga agagtccagg atctccagga 1080
cctggctcta caacctcgat aagaacctcg acaacgaata catctccacc ctcaactacc 1140
tctacgacag gatcaccaat gagctgacca actccttctc caagaactcc gccgccaacg 1200
tgaactatat tgccgaaact ctgggaatca accctgccga attcgccgaa caatatttca 1260
gattcagcat tatgaaagag cagaaaaacc tcggattcaa tatcaccaag ctcagggaag 1320
tgatgctgga caggaaggat atgtccgaga tcaggaaaaa tcataaggtg ttcgactcca 1380
tcaggaccaa ggtctacacc atgatggact ttgtgattta taggtattac atcgaagagg 1440
atgccaaggt ggctgccgcc aataagtccc tccccgataa tgagaagtcc ctgagcgaga 1500
aggatatctt tgtgattaac ctgaggggct ccttcaacga cgaccagaag gatgccctct 1560
actacgatga agctaataga atttggagaa agctcgaaaa tatcatgcac aacatcaagg 1620
aatttagggg aaacaagaca agagagtata agaagaagga cgcccctaga ctgcccagaa 1680
tcctgcccgc tggccgtgat gtttccgcct tcagcaaact catgtatgcc ctgaccatgt 1740
tcctggatgg caaggagatc aacgacctcc tgaccaccct gattaataaa ttcgataaca 1800
tccagagctt cctgaaggtg atgcctctca tcggagtcaa cgctaagttc gtggaggaat 1860
acgccttttt caaagactcc gccaagatcg ccgatgagct gaggctgatc aagtccttcg 1920
ctagaatggg agaacctatt gccgatgcca ggagggccat gtatatcgac gccatccgta 1980
ttttaggaac caacctgtcc tatgatgagc tcaaggccct cgccgacacc ttttccctgg 2040
acgagaacgg aaacaagctc aagaaaggca agcacggcat gagaaatttc attattaata 2100
acgtgatcag caataaaagg ttccactacc tgatcagata cggtgatcct gcccacctcc 2160
atgagatcgc caaaaacgag gccgtggtga agttcgtgct cggcaggatc gctgacatcc 2220
agaaaaaaca gggccagaac ggcaagaacc agatcgacag gtactacgaa acttgtatcg 2280
gaaaggataa gggcaagagc gtgagcgaaa aggtggacgc tctcacaaag atcatcaccg 2340
gaatgaacta cgaccaattc gacaagaaaa ggagcgtcat tgaggacacc ggcagggaaa 2400
acgccgagag ggagaagttt aaaaagatca tcagcctgta cctcaccgtg atctaccaca 2460
tcctcaagaa tattgtcaat atcaacgcca ggtacgtcat cggattccat tgcgtcgagc 2520
gtgatgctca actgtacaag gagaaaggct acgacatcaa tctcaagaaa ctggaagaga 2580
agggattcag ctccgtcacc aagctctgcg ctggcattga tgaaactgcc cccgataaga 2640
gaaaggacgt ggaaaaggag atggctgaaa gagccaagga gagcattgac agcctcgaga 2700
gcgccaaccc caagctgtat gccaattaca tcaaatacag cgacgagaag aaagccgagg 2760
agttcaccag gcagattaac agggagaagg ccaaaaccgc cctgaacgcc tacctgagga 2820
acaccaagtg gaatgtgatc atcagggagg acctcctgag aattgacaac aagacatgta 2880
ccctgttcag aaacaaggcc gtccacctgg aagtggccag gtatgtccac gcctatatca 2940
acgacattgc cgaggtcaat tcctacttcc aactgtacca ttacatcatg cagagaatta 3000
tcatgaatga gaggtacgag aaaagcagcg gaaaggtgtc cgagtacttc gacgctgtga 3060
atgacgagaa gaagtacaac gataggctcc tgaaactgct gtgtgtgcct ttcggctact 3120
gtatccccag gtttaagaac ctgagcatcg aggccctgtt cgataggaac gaggccgcca 3180
agttcgacaa ggagaaaaag aaggtgtccg gcaattccgg atccgactac aaagaccatg 3240
acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag ggtagcggca 3300
gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag 3360
tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg 3420
acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca 3480
agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg 3540
tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc 3600
acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca 3660
aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga 3720
accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc 3780
tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca 3840
tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc 3900
actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc 3960
tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc 4020
tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaattcg 4080
aagcccctct ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg 4140
gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc 4200
ccggaaacct ggccctgtct ttttgacgag cattcctagg ggtctttccc ctctcgccaa 4260
aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgagg 4320
acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg 4380
cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg 4440
ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa 4500
caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg 4560
gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca 4620
cggggacgtg gttttccttt gaaaaacacg atgataagct tgccaatgac cgagtacaag 4680
cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc 4740
gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg 4800
gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg 4860
gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg 4920
gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg 4980
cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg 5040
gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc 5100
cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc 5160
cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc 5220
gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgataagc ggccgcaatc 5280
aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt 5340
ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg 5400
ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc 5460
ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt 5520
ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg 5580
ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg 5640
gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct 5700
gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc 5760
cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtctccgcc 5820
ttcgccctca gacgagtcgg atctcccttt ggccgcctcc ccgcctggta cctttaagac 5880
caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg 5940
aagggctaat tcactcccaa cgaaaataag atctgctttt tgcttgtact gggtctctct 6000
ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc 6060
ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg 6120
gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt 6180
catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga 6240
ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 6300
caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 6360
cttatcatgt ctggctctag ctatcccgcc cctaactccg cccagttccg cccattctcc 6420
gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga 6480
gctattccag aagtagtgag gaggcttttt tggaggccta gacttttgca gagacggccc 6540
aaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 6600
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 6660
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 6720
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 6780
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 6840
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 6900
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6960
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7020
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 7080
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 7140
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 7200
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 7260
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7320
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7380
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7440
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 7500
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 7560
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 7620
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 7680
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 7740
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 7800
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 7860
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 7920
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 7980
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 8040
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 8100
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 8160
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 8220
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 8280
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 8340
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 8400
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 8460
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 8520
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 8580
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 8640
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 8700
gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 8760
cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 8820
gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 8880
gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 8940
gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 9000
ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 9060
gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 9120
ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct 9180
gacgcgtgta gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc 9240
aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt 9300
acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg 9360
aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacaat aaacgggtct 9420
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 9480
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 9540
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 9600
gcccgaacag ggacctgaaa gcgaaaggga aaccagagct ctctcgacgc aggactcggc 9660
ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 9720
tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 9780
gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 9840
taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 9900
tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 9960
gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 10020
ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 10080
gtaagaccac cgcacagcaa gcggccactg atcttcagac ctggaggagg agatatgagg 10140
gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta 10200
gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga 10260
gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc ctcaatgacg 10320
ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 10380
agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 10440
caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 10500
ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat 10560
aaatctctgg aacagattgg aatcacacga cctggatgga gtgggacaga gaaattaaca 10620
attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg 10680
aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa 10740
attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa 10800
tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt 10860
ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag 10920
gtggagagag agacagagac agatccattc gattagtgaa cggatctcga cggttaactt 10980
ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag 11040
caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttatcg 11100
atactagtat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 11160
cgctagcgag ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt 11220
tagagagata attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg 11280
acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga 11340
ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg 11400
gaaaggacga aacaccgaac ccctaccaac tggtcggggt ttgaaac 11447
<210> 2
<211> 11513
<212> DNA
<213> Linearized CasRx Vector，From pCasRxN-L1
<400> 2
tttttttgaa ttcgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 60
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 120
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 180
cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 240
tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag 300
aagattctag agccaccatg agccccaaga agaagagaaa ggtggaggcc agcatcgaaa 360
aaaaaaagtc cttcgccaag ggcatgggcg tgaagtccac actcgtgtcc ggctccaaag 420
tgtacatgac aaccttcgcc gaaggcagcg acgccaggct ggaaaagatc gtggagggcg 480
acagcatcag gagcgtgaat gagggcgagg ccttcagcgc tgaaatggcc gataaaaacg 540
ccggctataa gatcggcaac gccaaattca gccatcctaa gggctacgcc gtggtggcta 600
acaaccctct gtatacagga cccgtccagc aggatatgct cggcctgaag gaaactctgg 660
aaaagaggta cttcggcgag agcgctgatg gcaatgacaa tatttgtatc caggtgatcc 720
ataacatcct ggacattgaa aaaatcctcg ccgaatacat taccaacgcc gcctacgccg 780
tcaacaatat ctccggcctg gataaggaca ttattggatt cggcaagttc tccacagtgt 840
atacctacga cgaattcaaa gaccccgagc accatagggc cgctttcaac aataacgata 900
agctcatcaa cgccatcaag gcccagtatg acgagttcga caacttcctc gataacccca 960
gactcggcta tttcggccag gcctttttca gcaaggaggg cagaaattac atcatcaatt 1020
acggcaacga atgctatgac attctggccc tcctgagcgg actgaggcac tgggtggtcc 1080
ataacaacga agaagagtcc aggatctcca ggacctggct ctacaacctc gataagaacc 1140
tcgacaacga atacatctcc accctcaact acctctacga caggatcacc aatgagctga 1200
ccaactcctt ctccaagaac tccgccgcca acgtgaacta tattgccgaa actctgggaa 1260
tcaaccctgc cgaattcgcc gaacaatatt tcagattcag cattatgaaa gagcagaaaa 1320
acctcggatt caatatcacc aagctcaggg aagtgatgct ggacaggaag gatatgtccg 1380
agatcaggaa aaatcataag gtgttcgact ccatcaggac caaggtctac accatgatgg 1440
actttgtgat ttataggtat tacatcgaag aggatgccaa ggtggctgcc gccaataagt 1500
ccctccccga taatgagaag tccctgagcg agaaggatat ctttgtgatt aacctgaggg 1560
gctccttcaa cgacgaccag aaggatgccc tctactacga tgaagctaat agaatttgga 1620
gaaagctcga aaatatcatg cacaacatca aggaatttag gggaaacaag acaagagagt 1680
ataagaagaa ggacgcccct agactgccca gaatcctgcc cgctggccgt gatgtttccg 1740
ccttcagcaa actcatgtat gccctgacca tgttcctgga tggcaaggag atcaacgacc 1800
tcctgaccac cctgattaat aaattcgata acatccagag cttcctgaag gtgatgcctc 1860
tcatcggagt caacgctaag ttcgtggagg aatacgcctt tttcaaagac tccgccaaga 1920
tcgccgatga gctgaggctg atcaagtcct tcgctagaat gggagaacct attgccgatg 1980
ccaggagggc catgtatatc gacgccatcc gtattttagg aaccaacctg tcctatgatg 2040
agctcaaggc cctcgccgac accttttccc tggacgagaa cggaaacaag ctcaagaaag 2100
gcaagcacgg catgagaaat ttcattatta ataacgtgat cagcaataaa aggttccact 2160
acctgatcag atacggtgat cctgcccacc tccatgagat cgccaaaaac gaggccgtgg 2220
tgaagttcgt gctcggcagg atcgctgaca tccagaaaaa acagggccag aacggcaaga 2280
accagatcga caggtactac gaaacttgta tcggaaagga taagggcaag agcgtgagcg 2340
aaaaggtgga cgctctcaca aagatcatca ccggaatgaa ctacgaccaa ttcgacaaga 2400
aaaggagcgt cattgaggac accggcaggg aaaacgccga gagggagaag tttaaaaaga 2460
tcatcagcct gtacctcacc gtgatctacc acatcctcaa gaatattgtc aatatcaacg 2520
ccaggtacgt catcggattc cattgcgtcg agcgtgatgc tcaactgtac aaggagaaag 2580
gctacgacat caatctcaag aaactggaag agaagggatt cagctccgtc accaagctct 2640
gcgctggcat tgatgaaact gcccccgata agagaaagga cgtggaaaag gagatggctg 2700
aaagagccaa ggagagcatt gacagcctcg agagcgccaa ccccaagctg tatgccaatt 2760
acatcaaata cagcgacgag aagaaagccg aggagttcac caggcagatt aacagggaga 2820
aggccaaaac cgccctgaac gcctacctga ggaacaccaa gtggaatgtg atcatcaggg 2880
aggacctcct gagaattgac aacaagacat gtaccctgtt cagaaacaag gccgtccacc 2940
tggaagtggc caggtatgtc cacgcctata tcaacgacat tgccgaggtc aattcctact 3000
tccaactgta ccattacatc atgcagagaa ttatcatgaa tgagaggtac gagaaaagca 3060
gcggaaaggt gtccgagtac ttcgacgctg tgaatgacga gaagaagtac aacgataggc 3120
tcctgaaact gctgtgtgtg cctttcggct actgtatccc caggtttaag aacctgagca 3180
tcgaggccct gttcgatagg aacgaggccg ccaagttcga caaggagaaa aagaaggtgt 3240
ccggcaattc cggatccgga cctaagaaaa agaggaaggt ggcggcggct gactacaaag 3300
accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaagggta 3360
gcggcagtgg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg 3420
gcccagtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 3480
acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 3540
acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 3600
ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 3660
agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 3720
tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 3780
tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 3840
acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 3900
acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 3960
ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 4020
actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 4080
tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt 4140
aattcgaagc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata 4200
aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt 4260
gagggcccgg aaacctggcc ctgtcttttt gacgagcatt cctaggggtc tttcccctct 4320
cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc 4380
ttgaggacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga 4440
caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc 4500
ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt 4560
attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg 4620
gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc 4680
gaaccacggg gacgtggttt tcctttgaaa aacacgatga taagcttgcc aatgaccgag 4740
tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc 4800
gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc 4860
gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag 4920
gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa 4980
gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg 5040
gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg 5100
ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc 5160
gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc 5220
gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag 5280
gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg ataagcggcc 5340
gcaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg 5400
ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc 5460
gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt 5520
tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca 5580
ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc 5640
ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc 5700
tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc 5760
tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc 5820
tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc 5880
tccgccttcg ccctcagacg agtcggatct ccctttggcc gcctccccgc ctggtacctt 5940
taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg 6000
gactggaagg gctaattcac tcccaacgaa aataagatct gctttttgct tgtactgggt 6060
ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 6120
ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 6180
actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta 6240
gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga 6300
gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 6360
atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 6420
atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca gttccgccca 6480
ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 6540
ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctagact tttgcagaga 6600
cggcccaaat tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 6660
aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 6720
gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 6780
gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 6840
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 6900
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 6960
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 7020
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 7080
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 7140
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 7200
aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 7260
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 7320
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 7380
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 7440
gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 7500
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 7560
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 7620
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 7680
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 7740
taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 7800
tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 7860
cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 7920
gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 7980
cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 8040
ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 8100
aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 8160
atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 8220
tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 8280
gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 8340
aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 8400
acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 8460
ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 8520
tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 8580
aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 8640
catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 8700
atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 8760
aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 8820
gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca 8880
catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc 8940
ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc 9000
agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 9060
gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg 9120
atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg 9180
attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtgc 9240
caagctgacg cgtgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga 9300
gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa 9360
ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa 9420
ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga tacaataaac 9480
gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 9540
ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 9600
tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 9660
agtggcgccc gaacagggac ctgaaagcga aagggaaacc agagctctct cgacgcagga 9720
ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa 9780
aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc 9840
gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat 9900
ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg 9960
gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc 10020
agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc 10080
atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa 10140
acaaaagtaa gaccaccgca cagcaagcgg ccactgatct tcagacctgg aggaggagat 10200
atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 10260
ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 10320
ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcctca 10380
atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 10440
ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 10500
cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 10560
atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 10620
agtaataaat ctctggaaca gattggaatc acacgacctg gatggagtgg gacagagaaa 10680
ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa 10740
agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca 10800
taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt 10860
taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat 10920
tatcgtttca gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag 10980
aagaaggtgg agagagagac agagacagat ccattcgatt agtgaacgga tctcgacggt 11040
taacttttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca 11100
taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt 11160
ttatcgatac tagtattatg cccagtacat gaccttatgg gactttccta cttggcagta 11220
catctacgct agcgagggcc tatttcccat gattccttca tatttgcata tacgatacaa 11280
ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa 11340
tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa 11400
aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat 11460
cttgtggaaa ggacgaaaca ccgaacccct accaactggt cggggtttga aac 11513
<210> 3
<211> 6911
<212> DNA
<213> Linearized CasRx Vector，From pCasRx-A1
<400> 3
tttttttgaa ttcgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 60
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 120
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 180
cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 240
tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag 300
aagattctag agccaccatg atcgaaaaaa aaaagtcctt cgccaagggc atgggcgtga 360
agtccacact cgtgtccggc tccaaagtgt acatgacaac cttcgccgaa ggcagcgacg 420
ccaggctgga aaagatcgtg gagggcgaca gcatcaggag cgtgaatgag ggcgaggcct 480
tcagcgctga aatggccgat aaaaacgccg gctataagat cggcaacgcc aaattcagcc 540
atcctaaggg ctacgccgtg gtggctaaca accctctgta tacaggaccc gtccagcagg 600
atatgctcgg cctgaaggaa actctggaaa agaggtactt cggcgagagc gctgatggca 660
atgacaatat ttgtatccag gtgatccata acatcctgga cattgaaaaa atcctcgccg 720
aatacattac caacgccgcc tacgccgtca acaatatctc cggcctggat aaggacatta 780
ttggattcgg caagttctcc acagtgtata cctacgacga attcaaagac cccgagcacc 840
atagggccgc tttcaacaat aacgataagc tcatcaacgc catcaaggcc cagtatgacg 900
agttcgacaa cttcctcgat aaccccagac tcggctattt cggccaggcc tttttcagca 960
aggagggcag aaattacatc atcaattacg gcaacgaatg ctatgacatt ctggccctcc 1020
tgagcggact gaggcactgg gtggtccata acaacgaaga agagtccagg atctccagga 1080
cctggctcta caacctcgat aagaacctcg acaacgaata catctccacc ctcaactacc 1140
tctacgacag gatcaccaat gagctgacca actccttctc caagaactcc gccgccaacg 1200
tgaactatat tgccgaaact ctgggaatca accctgccga attcgccgaa caatatttca 1260
gattcagcat tatgaaagag cagaaaaacc tcggattcaa tatcaccaag ctcagggaag 1320
tgatgctgga caggaaggat atgtccgaga tcaggaaaaa tcataaggtg ttcgactcca 1380
tcaggaccaa ggtctacacc atgatggact ttgtgattta taggtattac atcgaagagg 1440
atgccaaggt ggctgccgcc aataagtccc tccccgataa tgagaagtcc ctgagcgaga 1500
aggatatctt tgtgattaac ctgaggggct ccttcaacga cgaccagaag gatgccctct 1560
actacgatga agctaataga atttggagaa agctcgaaaa tatcatgcac aacatcaagg 1620
aatttagggg aaacaagaca agagagtata agaagaagga cgcccctaga ctgcccagaa 1680
tcctgcccgc tggccgtgat gtttccgcct tcagcaaact catgtatgcc ctgaccatgt 1740
tcctggatgg caaggagatc aacgacctcc tgaccaccct gattaataaa ttcgataaca 1800
tccagagctt cctgaaggtg atgcctctca tcggagtcaa cgctaagttc gtggaggaat 1860
acgccttttt caaagactcc gccaagatcg ccgatgagct gaggctgatc aagtccttcg 1920
ctagaatggg agaacctatt gccgatgcca ggagggccat gtatatcgac gccatccgta 1980
ttttaggaac caacctgtcc tatgatgagc tcaaggccct cgccgacacc ttttccctgg 2040
acgagaacgg aaacaagctc aagaaaggca agcacggcat gagaaatttc attattaata 2100
acgtgatcag caataaaagg ttccactacc tgatcagata cggtgatcct gcccacctcc 2160
atgagatcgc caaaaacgag gccgtggtga agttcgtgct cggcaggatc gctgacatcc 2220
agaaaaaaca gggccagaac ggcaagaacc agatcgacag gtactacgaa acttgtatcg 2280
gaaaggataa gggcaagagc gtgagcgaaa aggtggacgc tctcacaaag atcatcaccg 2340
gaatgaacta cgaccaattc gacaagaaaa ggagcgtcat tgaggacacc ggcagggaaa 2400
acgccgagag ggagaagttt aaaaagatca tcagcctgta cctcaccgtg atctaccaca 2460
tcctcaagaa tattgtcaat atcaacgcca ggtacgtcat cggattccat tgcgtcgagc 2520
gtgatgctca actgtacaag gagaaaggct acgacatcaa tctcaagaaa ctggaagaga 2580
agggattcag ctccgtcacc aagctctgcg ctggcattga tgaaactgcc cccgataaga 2640
gaaaggacgt ggaaaaggag atggctgaaa gagccaagga gagcattgac agcctcgaga 2700
gcgccaaccc caagctgtat gccaattaca tcaaatacag cgacgagaag aaagccgagg 2760
agttcaccag gcagattaac agggagaagg ccaaaaccgc cctgaacgcc tacctgagga 2820
acaccaagtg gaatgtgatc atcagggagg acctcctgag aattgacaac aagacatgta 2880
ccctgttcag aaacaaggcc gtccacctgg aagtggccag gtatgtccac gcctatatca 2940
acgacattgc cgaggtcaat tcctacttcc aactgtacca ttacatcatg cagagaatta 3000
tcatgaatga gaggtacgag aaaagcagcg gaaaggtgtc cgagtacttc gacgctgtga 3060
atgacgagaa gaagtacaac gataggctcc tgaaactgct gtgtgtgcct ttcggctact 3120
gtatccccag gtttaagaac ctgagcatcg aggccctgtt cgataggaac gaggccgcca 3180
agttcgacaa ggagaaaaag aaggtgtccg gcaattccgg atccgactac aaagaccatg 3240
acggtgatta taaagatcat gacatcgatt acaaggatga cgatgacaag taataagcta 3300
gcataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 3360
tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 3420
ccgtatggct ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga 3480
actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 3540
ttccgtggtg tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccatctagct 3600
ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 3660
aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg 3720
ttttttaaag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 3780
tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 3840
ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 3900
ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 3960
tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 4020
gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 4080
ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 4140
cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 4200
tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 4260
ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt 4320
ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 4380
tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 4440
gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 4500
tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 4560
cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 4620
tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 4680
ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 4740
ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 4800
attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 4860
gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 4920
ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 4980
cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 5040
gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 5100
tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 5160
gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 5220
ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 5280
tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 5340
gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 5400
caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 5460
cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 5520
atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 5580
gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 5640
attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 5700
cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 5760
atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 5820
tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 5880
ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 5940
ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 6000
cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6060
gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6120
gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6180
acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6240
gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6300
agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6360
tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6420
agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtcctgc 6480
aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga cctttggtcg 6540
cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg 6600
ttcctgcggc cattcggtac aattcacgcg tgagggccta tttcccatga ttccttcata 6660
tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac 6720
aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt 6780
tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga 6840
tttcttggct ttatatatct tgtggaaagg acgaaacacc gaacccctac caactggtcg 6900
gggtttgaaa c 6911
<210> 4
<211> 6977
<212> DNA
<213> Linearized CasRx Vector，From pCasRxN-A1
<400> 4
tttttttgaa ttcgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 60
caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 120
caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 180
cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 240
tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag 300
aagattctag agccaccatg agccccaaga agaagagaaa ggtggaggcc agcatcgaaa 360
aaaaaaagtc cttcgccaag ggcatgggcg tgaagtccac actcgtgtcc ggctccaaag 420
tgtacatgac aaccttcgcc gaaggcagcg acgccaggct ggaaaagatc gtggagggcg 480
acagcatcag gagcgtgaat gagggcgagg ccttcagcgc tgaaatggcc gataaaaacg 540
ccggctataa gatcggcaac gccaaattca gccatcctaa gggctacgcc gtggtggcta 600
acaaccctct gtatacagga cccgtccagc aggatatgct cggcctgaag gaaactctgg 660
aaaagaggta cttcggcgag agcgctgatg gcaatgacaa tatttgtatc caggtgatcc 720
ataacatcct ggacattgaa aaaatcctcg ccgaatacat taccaacgcc gcctacgccg 780
tcaacaatat ctccggcctg gataaggaca ttattggatt cggcaagttc tccacagtgt 840
atacctacga cgaattcaaa gaccccgagc accatagggc cgctttcaac aataacgata 900
agctcatcaa cgccatcaag gcccagtatg acgagttcga caacttcctc gataacccca 960
gactcggcta tttcggccag gcctttttca gcaaggaggg cagaaattac atcatcaatt 1020
acggcaacga atgctatgac attctggccc tcctgagcgg actgaggcac tgggtggtcc 1080
ataacaacga agaagagtcc aggatctcca ggacctggct ctacaacctc gataagaacc 1140
tcgacaacga atacatctcc accctcaact acctctacga caggatcacc aatgagctga 1200
ccaactcctt ctccaagaac tccgccgcca acgtgaacta tattgccgaa actctgggaa 1260
tcaaccctgc cgaattcgcc gaacaatatt tcagattcag cattatgaaa gagcagaaaa 1320
acctcggatt caatatcacc aagctcaggg aagtgatgct ggacaggaag gatatgtccg 1380
agatcaggaa aaatcataag gtgttcgact ccatcaggac caaggtctac accatgatgg 1440
actttgtgat ttataggtat tacatcgaag aggatgccaa ggtggctgcc gccaataagt 1500
ccctccccga taatgagaag tccctgagcg agaaggatat ctttgtgatt aacctgaggg 1560
gctccttcaa cgacgaccag aaggatgccc tctactacga tgaagctaat agaatttgga 1620
gaaagctcga aaatatcatg cacaacatca aggaatttag gggaaacaag acaagagagt 1680
ataagaagaa ggacgcccct agactgccca gaatcctgcc cgctggccgt gatgtttccg 1740
ccttcagcaa actcatgtat gccctgacca tgttcctgga tggcaaggag atcaacgacc 1800
tcctgaccac cctgattaat aaattcgata acatccagag cttcctgaag gtgatgcctc 1860
tcatcggagt caacgctaag ttcgtggagg aatacgcctt tttcaaagac tccgccaaga 1920
tcgccgatga gctgaggctg atcaagtcct tcgctagaat gggagaacct attgccgatg 1980
ccaggagggc catgtatatc gacgccatcc gtattttagg aaccaacctg tcctatgatg 2040
agctcaaggc cctcgccgac accttttccc tggacgagaa cggaaacaag ctcaagaaag 2100
gcaagcacgg catgagaaat ttcattatta ataacgtgat cagcaataaa aggttccact 2160
acctgatcag atacggtgat cctgcccacc tccatgagat cgccaaaaac gaggccgtgg 2220
tgaagttcgt gctcggcagg atcgctgaca tccagaaaaa acagggccag aacggcaaga 2280
accagatcga caggtactac gaaacttgta tcggaaagga taagggcaag agcgtgagcg 2340
aaaaggtgga cgctctcaca aagatcatca ccggaatgaa ctacgaccaa ttcgacaaga 2400
aaaggagcgt cattgaggac accggcaggg aaaacgccga gagggagaag tttaaaaaga 2460
tcatcagcct gtacctcacc gtgatctacc acatcctcaa gaatattgtc aatatcaacg 2520
ccaggtacgt catcggattc cattgcgtcg agcgtgatgc tcaactgtac aaggagaaag 2580
gctacgacat caatctcaag aaactggaag agaagggatt cagctccgtc accaagctct 2640
gcgctggcat tgatgaaact gcccccgata agagaaagga cgtggaaaag gagatggctg 2700
aaagagccaa ggagagcatt gacagcctcg agagcgccaa ccccaagctg tatgccaatt 2760
acatcaaata cagcgacgag aagaaagccg aggagttcac caggcagatt aacagggaga 2820
aggccaaaac cgccctgaac gcctacctga ggaacaccaa gtggaatgtg atcatcaggg 2880
aggacctcct gagaattgac aacaagacat gtaccctgtt cagaaacaag gccgtccacc 2940
tggaagtggc caggtatgtc cacgcctata tcaacgacat tgccgaggtc aattcctact 3000
tccaactgta ccattacatc atgcagagaa ttatcatgaa tgagaggtac gagaaaagca 3060
gcggaaaggt gtccgagtac ttcgacgctg tgaatgacga gaagaagtac aacgataggc 3120
tcctgaaact gctgtgtgtg cctttcggct actgtatccc caggtttaag aacctgagca 3180
tcgaggccct gttcgatagg aacgaggccg ccaagttcga caaggagaaa aagaaggtgt 3240
ccggcaattc cggatccgga cctaagaaaa agaggaaggt ggcggcggct gactacaaag 3300
accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat gacaagtaat 3360
aagctagcat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa 3420
ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat 3480
tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttag ttcttgccac 3540
ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 3600
tgacaattcc gtggtgttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 3660
ctagctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 3720
ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggagatgt 3780
gggaggtttt ttaaagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct 3840
gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 3900
ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta 3960
ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac 4020
gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 4080
acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 4140
ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 4200
gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca 4260
tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 4320
ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa 4380
gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 4440
gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc 4500
tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 4560
cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 4620
atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata 4680
cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 4740
tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 4800
tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 4860
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 4920
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 4980
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 5040
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 5100
cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 5160
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 5220
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 5280
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 5340
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 5400
cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 5460
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 5520
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 5580
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 5640
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 5700
tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 5760
ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 5820
accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 5880
aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 5940
ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 6000
gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 6060
ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 6120
ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 6180
ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 6240
gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 6300
cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 6360
cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 6420
cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 6480
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 6540
tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt 6600
tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac 6660
taggggttcc tgcggccatt cggtacaatt cacgcgtgag ggcctatttc ccatgattcc 6720
ttcatatttg catatacgat acaaggctgt tagagagata attggaatta atttgactgt 6780
aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt 6840
tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta 6900
tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgaac ccctaccaac 6960
tggtcggggt ttgaaac 6977
<210> 5
<211> 24
<212> DNA
<213> U6 Primer
<400> 5
atggactatc atatgcttac cgta 24
<210> 6
<211> 11469
<212> DNA
<213> gRNA-NC Vector，pCasRx-L1
<400> 6
acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60
acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120
cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180
attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240
tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300
agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360
ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420
cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480
tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540
gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600
aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660
aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720
agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780
atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840
gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900
taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960
acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020
cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080
ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140
tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200
gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260
aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320
gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380
aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440
ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500
acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560
ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680
tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740
tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggttaacttt 1800
taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc 1860
aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttatcga 1920
tactagtatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 1980
gctagcgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 2040
agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 2100
cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 2160
tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 2220
aaaggacgaa acaccgaacc cctaccaact ggtcggggtt tgaaactcac cagaagcgta 2280
ccatactctt tttttgaatt cgtattagtc atcgctatta ccatggtgat gcggttttgg 2340
cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 2400
attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 2460
aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 2520
agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac 2580
ctccatagaa gattctagag ccaccatgat cgaaaaaaaa aagtccttcg ccaagggcat 2640
gggcgtgaag tccacactcg tgtccggctc caaagtgtac atgacaacct tcgccgaagg 2700
cagcgacgcc aggctggaaa agatcgtgga gggcgacagc atcaggagcg tgaatgaggg 2760
cgaggccttc agcgctgaaa tggccgataa aaacgccggc tataagatcg gcaacgccaa 2820
attcagccat cctaagggct acgccgtggt ggctaacaac cctctgtata caggacccgt 2880
ccagcaggat atgctcggcc tgaaggaaac tctggaaaag aggtacttcg gcgagagcgc 2940
tgatggcaat gacaatattt gtatccaggt gatccataac atcctggaca ttgaaaaaat 3000
cctcgccgaa tacattacca acgccgccta cgccgtcaac aatatctccg gcctggataa 3060
ggacattatt ggattcggca agttctccac agtgtatacc tacgacgaat tcaaagaccc 3120
cgagcaccat agggccgctt tcaacaataa cgataagctc atcaacgcca tcaaggccca 3180
gtatgacgag ttcgacaact tcctcgataa ccccagactc ggctatttcg gccaggcctt 3240
tttcagcaag gagggcagaa attacatcat caattacggc aacgaatgct atgacattct 3300
ggccctcctg agcggactga ggcactgggt ggtccataac aacgaagaag agtccaggat 3360
ctccaggacc tggctctaca acctcgataa gaacctcgac aacgaataca tctccaccct 3420
caactacctc tacgacagga tcaccaatga gctgaccaac tccttctcca agaactccgc 3480
cgccaacgtg aactatattg ccgaaactct gggaatcaac cctgccgaat tcgccgaaca 3540
atatttcaga ttcagcatta tgaaagagca gaaaaacctc ggattcaata tcaccaagct 3600
cagggaagtg atgctggaca ggaaggatat gtccgagatc aggaaaaatc ataaggtgtt 3660
cgactccatc aggaccaagg tctacaccat gatggacttt gtgatttata ggtattacat 3720
cgaagaggat gccaaggtgg ctgccgccaa taagtccctc cccgataatg agaagtccct 3780
gagcgagaag gatatctttg tgattaacct gaggggctcc ttcaacgacg accagaagga 3840
tgccctctac tacgatgaag ctaatagaat ttggagaaag ctcgaaaata tcatgcacaa 3900
catcaaggaa tttaggggaa acaagacaag agagtataag aagaaggacg cccctagact 3960
gcccagaatc ctgcccgctg gccgtgatgt ttccgccttc agcaaactca tgtatgccct 4020
gaccatgttc ctggatggca aggagatcaa cgacctcctg accaccctga ttaataaatt 4080
cgataacatc cagagcttcc tgaaggtgat gcctctcatc ggagtcaacg ctaagttcgt 4140
ggaggaatac gcctttttca aagactccgc caagatcgcc gatgagctga ggctgatcaa 4200
gtccttcgct agaatgggag aacctattgc cgatgccagg agggccatgt atatcgacgc 4260
catccgtatt ttaggaacca acctgtccta tgatgagctc aaggccctcg ccgacacctt 4320
ttccctggac gagaacggaa acaagctcaa gaaaggcaag cacggcatga gaaatttcat 4380
tattaataac gtgatcagca ataaaaggtt ccactacctg atcagatacg gtgatcctgc 4440
ccacctccat gagatcgcca aaaacgaggc cgtggtgaag ttcgtgctcg gcaggatcgc 4500
tgacatccag aaaaaacagg gccagaacgg caagaaccag atcgacaggt actacgaaac 4560
ttgtatcgga aaggataagg gcaagagcgt gagcgaaaag gtggacgctc tcacaaagat 4620
catcaccgga atgaactacg accaattcga caagaaaagg agcgtcattg aggacaccgg 4680
cagggaaaac gccgagaggg agaagtttaa aaagatcatc agcctgtacc tcaccgtgat 4740
ctaccacatc ctcaagaata ttgtcaatat caacgccagg tacgtcatcg gattccattg 4800
cgtcgagcgt gatgctcaac tgtacaagga gaaaggctac gacatcaatc tcaagaaact 4860
ggaagagaag ggattcagct ccgtcaccaa gctctgcgct ggcattgatg aaactgcccc 4920
cgataagaga aaggacgtgg aaaaggagat ggctgaaaga gccaaggaga gcattgacag 4980
cctcgagagc gccaacccca agctgtatgc caattacatc aaatacagcg acgagaagaa 5040
agccgaggag ttcaccaggc agattaacag ggagaaggcc aaaaccgccc tgaacgccta 5100
cctgaggaac accaagtgga atgtgatcat cagggaggac ctcctgagaa ttgacaacaa 5160
gacatgtacc ctgttcagaa acaaggccgt ccacctggaa gtggccaggt atgtccacgc 5220
ctatatcaac gacattgccg aggtcaattc ctacttccaa ctgtaccatt acatcatgca 5280
gagaattatc atgaatgaga ggtacgagaa aagcagcgga aaggtgtccg agtacttcga 5340
cgctgtgaat gacgagaaga agtacaacga taggctcctg aaactgctgt gtgtgccttt 5400
cggctactgt atccccaggt ttaagaacct gagcatcgag gccctgttcg ataggaacga 5460
ggccgccaag ttcgacaagg agaaaaagaa ggtgtccggc aattccggat ccgactacaa 5520
agaccatgac ggtgattata aagatcatga catcgattac aaggatgacg atgacaaggg 5580
tagcggcagt ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc 5640
tggcccagtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 5700
ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 5760
ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 5820
caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat 5880
gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 5940
cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 6000
cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 6060
gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 6120
gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 6180
cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 6240
ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 6300
ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 6360
gtaattcgaa gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa 6420
taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat 6480
gtgagggccc ggaaacctgg ccctgtcttt ttgacgagca ttcctagggg tctttcccct 6540
ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct 6600
tcttgaggac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc 6660
gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa 6720
ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc 6780
gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg 6840
gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc 6900
ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagcttg ccaatgaccg 6960
agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 7020
tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca 7080
tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 7140
aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg 7200
aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 7260
tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt 7320
ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg 7380
tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct 7440
ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg 7500
aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgataagcgg 7560
ccgcaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 7620
tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 7680
ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga 7740
gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc 7800
cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct 7860
ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 7920
gctgttgggc actgacaatt ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct 7980
gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc 8040
cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg 8100
tctccgcctt cgccctcaga cgagtcggat ctccctttgg ccgcctcccc gcctggtacc 8160
tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg 8220
gggactggaa gggctaattc actcccaacg aaaataagat ctgctttttg cttgtactgg 8280
gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 8340
gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 8400
tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag 8460
tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga 8520
gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 8580
aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 8640
caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc 8700
cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg 8760
gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctaga cttttgcaga 8820
gacggcccaa attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 8880
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 8940
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 9000
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 9060
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 9120
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 9180
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 9240
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 9300
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 9360
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 9420
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 9480
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 9540
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 9600
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 9660
tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 9720
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 9780
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 9840
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 9900
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 9960
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 10020
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 10080
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 10140
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 10200
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 10260
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 10320
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 10380
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 10440
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 10500
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 10560
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 10620
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 10680
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 10740
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 10800
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 10860
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 10920
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 10980
cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 11040
aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 11100
cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 11160
gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 11220
tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 11280
aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg 11340
cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg 11400
cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt 11460
gccaagctg 11469
<210> 7
<211> 11535
<212> DNA
<213> gRNA-NC Vector，pCasRxN-L1
<400> 7
acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60
acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120
cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180
attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240
tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300
agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360
ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420
cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480
tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540
gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600
aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660
aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720
agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780
atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840
gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900
taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960
acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020
cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080
ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140
tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200
gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260
aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320
gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380
aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440
ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500
acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560
ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680
tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740
tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggttaacttt 1800
taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc 1860
aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttatcga 1920
tactagtatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac 1980
gctagcgagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt 2040
agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga 2100
cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac 2160
tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg 2220
aaaggacgaa acaccgaacc cctaccaact ggtcggggtt tgaaactcac cagaagcgta 2280
ccatactctt tttttgaatt cgtattagtc atcgctatta ccatggtgat gcggttttgg 2340
cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc 2400
attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt 2460
aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 2520
agcagagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg ctgttttgac 2580
ctccatagaa gattctagag ccaccatgag ccccaagaag aagagaaagg tggaggccag 2640
catcgaaaaa aaaaagtcct tcgccaaggg catgggcgtg aagtccacac tcgtgtccgg 2700
ctccaaagtg tacatgacaa ccttcgccga aggcagcgac gccaggctgg aaaagatcgt 2760
ggagggcgac agcatcagga gcgtgaatga gggcgaggcc ttcagcgctg aaatggccga 2820
taaaaacgcc ggctataaga tcggcaacgc caaattcagc catcctaagg gctacgccgt 2880
ggtggctaac aaccctctgt atacaggacc cgtccagcag gatatgctcg gcctgaagga 2940
aactctggaa aagaggtact tcggcgagag cgctgatggc aatgacaata tttgtatcca 3000
ggtgatccat aacatcctgg acattgaaaa aatcctcgcc gaatacatta ccaacgccgc 3060
ctacgccgtc aacaatatct ccggcctgga taaggacatt attggattcg gcaagttctc 3120
cacagtgtat acctacgacg aattcaaaga ccccgagcac catagggccg ctttcaacaa 3180
taacgataag ctcatcaacg ccatcaaggc ccagtatgac gagttcgaca acttcctcga 3240
taaccccaga ctcggctatt tcggccaggc ctttttcagc aaggagggca gaaattacat 3300
catcaattac ggcaacgaat gctatgacat tctggccctc ctgagcggac tgaggcactg 3360
ggtggtccat aacaacgaag aagagtccag gatctccagg acctggctct acaacctcga 3420
taagaacctc gacaacgaat acatctccac cctcaactac ctctacgaca ggatcaccaa 3480
tgagctgacc aactccttct ccaagaactc cgccgccaac gtgaactata ttgccgaaac 3540
tctgggaatc aaccctgccg aattcgccga acaatatttc agattcagca ttatgaaaga 3600
gcagaaaaac ctcggattca atatcaccaa gctcagggaa gtgatgctgg acaggaagga 3660
tatgtccgag atcaggaaaa atcataaggt gttcgactcc atcaggacca aggtctacac 3720
catgatggac tttgtgattt ataggtatta catcgaagag gatgccaagg tggctgccgc 3780
caataagtcc ctccccgata atgagaagtc cctgagcgag aaggatatct ttgtgattaa 3840
cctgaggggc tccttcaacg acgaccagaa ggatgccctc tactacgatg aagctaatag 3900
aatttggaga aagctcgaaa atatcatgca caacatcaag gaatttaggg gaaacaagac 3960
aagagagtat aagaagaagg acgcccctag actgcccaga atcctgcccg ctggccgtga 4020
tgtttccgcc ttcagcaaac tcatgtatgc cctgaccatg ttcctggatg gcaaggagat 4080
caacgacctc ctgaccaccc tgattaataa attcgataac atccagagct tcctgaaggt 4140
gatgcctctc atcggagtca acgctaagtt cgtggaggaa tacgcctttt tcaaagactc 4200
cgccaagatc gccgatgagc tgaggctgat caagtccttc gctagaatgg gagaacctat 4260
tgccgatgcc aggagggcca tgtatatcga cgccatccgt attttaggaa ccaacctgtc 4320
ctatgatgag ctcaaggccc tcgccgacac cttttccctg gacgagaacg gaaacaagct 4380
caagaaaggc aagcacggca tgagaaattt cattattaat aacgtgatca gcaataaaag 4440
gttccactac ctgatcagat acggtgatcc tgcccacctc catgagatcg ccaaaaacga 4500
ggccgtggtg aagttcgtgc tcggcaggat cgctgacatc cagaaaaaac agggccagaa 4560
cggcaagaac cagatcgaca ggtactacga aacttgtatc ggaaaggata agggcaagag 4620
cgtgagcgaa aaggtggacg ctctcacaaa gatcatcacc ggaatgaact acgaccaatt 4680
cgacaagaaa aggagcgtca ttgaggacac cggcagggaa aacgccgaga gggagaagtt 4740
taaaaagatc atcagcctgt acctcaccgt gatctaccac atcctcaaga atattgtcaa 4800
tatcaacgcc aggtacgtca tcggattcca ttgcgtcgag cgtgatgctc aactgtacaa 4860
ggagaaaggc tacgacatca atctcaagaa actggaagag aagggattca gctccgtcac 4920
caagctctgc gctggcattg atgaaactgc ccccgataag agaaaggacg tggaaaagga 4980
gatggctgaa agagccaagg agagcattga cagcctcgag agcgccaacc ccaagctgta 5040
tgccaattac atcaaataca gcgacgagaa gaaagccgag gagttcacca ggcagattaa 5100
cagggagaag gccaaaaccg ccctgaacgc ctacctgagg aacaccaagt ggaatgtgat 5160
catcagggag gacctcctga gaattgacaa caagacatgt accctgttca gaaacaaggc 5220
cgtccacctg gaagtggcca ggtatgtcca cgcctatatc aacgacattg ccgaggtcaa 5280
ttcctacttc caactgtacc attacatcat gcagagaatt atcatgaatg agaggtacga 5340
gaaaagcagc ggaaaggtgt ccgagtactt cgacgctgtg aatgacgaga agaagtacaa 5400
cgataggctc ctgaaactgc tgtgtgtgcc tttcggctac tgtatcccca ggtttaagaa 5460
cctgagcatc gaggccctgt tcgataggaa cgaggccgcc aagttcgaca aggagaaaaa 5520
gaaggtgtcc ggcaattccg gatccggacc taagaaaaag aggaaggtgg cggcggctga 5580
ctacaaagac catgacggtg attataaaga tcatgacatc gattacaagg atgacgatga 5640
caagggtagc ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga 5700
gaatcctggc ccagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt 5760
cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga 5820
tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc 5880
ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga 5940
ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg 6000
caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg 6060
cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat 6120
cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa 6180
gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt 6240
gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc 6300
cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga 6360
tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct 6420
gtacaagtaa ttcgaagccc ctctccctcc ccccccccta acgttactgg ccgaagccgc 6480
ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt 6540
ggcaatgtga gggcccggaa acctggccct gtctttttga cgagcattcc taggggtctt 6600
tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg 6660
gaagcttctt gaggacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca 6720
cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg 6780
gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc 6840
tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct 6900
gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta 6960
ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata agcttgccaa 7020
tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac 7080
gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc 7140
gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca 7200
tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga 7260
gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt 7320
cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc 7380
ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca 7440
gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg 7500
agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg 7560
acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgat 7620
aagcggccgc aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa 7680
ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat 7740
tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta 7800
tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc 7860
aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt 7920
ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg 7980
ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc 8040
atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc 8100
ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct 8160
tccgcgtctc cgccttcgcc ctcagacgag tcggatctcc ctttggccgc ctccccgcct 8220
ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga 8280
aaagggggga ctggaagggc taattcactc ccaacgaaaa taagatctgc tttttgcttg 8340
tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 8400
cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 8460
gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 8520
tagcagtagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat 8580
atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag 8640
catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 8700
actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccagt 8760
tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 8820
gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctagacttt 8880
tgcagagacg gcccaaattc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 8940
ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 9000
taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 9060
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 9120
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 9180
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 9240
gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 9300
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 9360
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 9420
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 9480
ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 9540
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 9600
ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 9660
gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 9720
aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 9780
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 9840
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 9900
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 9960
gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 10020
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 10080
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 10140
ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 10200
atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 10260
ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 10320
tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 10380
attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 10440
tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 10500
ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 10560
gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 10620
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 10680
gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 10740
aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 10800
taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 10860
tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 10920
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 10980
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 11040
tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat 11100
aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac 11160
ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 11220
agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat 11280
gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga 11340
tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg 11400
gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct 11460
gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 11520
gccagtgcca agctg 11535
<210> 8
<211> 6933
<212> DNA
<213> gRNA-NC Vector，pCasRx-A1
<400> 8
gcggccattc ggtacaattc acgcgtgagg gcctatttcc catgattcct tcatatttgc 60
atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga 120
tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa 180
aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct 240
tggctttata tatcttgtgg aaaggacgaa acaccgaacc cctaccaact ggtcggggtt 300
tgaaactcac cagaagcgta ccatactctt tttttgaatt cgtattagtc atcgctatta 360
ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 420
gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 480
gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 540
tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 600
gccatccacg ctgttttgac ctccatagaa gattctagag ccaccatgat cgaaaaaaaa 660
aagtccttcg ccaagggcat gggcgtgaag tccacactcg tgtccggctc caaagtgtac 720
atgacaacct tcgccgaagg cagcgacgcc aggctggaaa agatcgtgga gggcgacagc 780
atcaggagcg tgaatgaggg cgaggccttc agcgctgaaa tggccgataa aaacgccggc 840
tataagatcg gcaacgccaa attcagccat cctaagggct acgccgtggt ggctaacaac 900
cctctgtata caggacccgt ccagcaggat atgctcggcc tgaaggaaac tctggaaaag 960
aggtacttcg gcgagagcgc tgatggcaat gacaatattt gtatccaggt gatccataac 1020
atcctggaca ttgaaaaaat cctcgccgaa tacattacca acgccgccta cgccgtcaac 1080
aatatctccg gcctggataa ggacattatt ggattcggca agttctccac agtgtatacc 1140
tacgacgaat tcaaagaccc cgagcaccat agggccgctt tcaacaataa cgataagctc 1200
atcaacgcca tcaaggccca gtatgacgag ttcgacaact tcctcgataa ccccagactc 1260
ggctatttcg gccaggcctt tttcagcaag gagggcagaa attacatcat caattacggc 1320
aacgaatgct atgacattct ggccctcctg agcggactga ggcactgggt ggtccataac 1380
aacgaagaag agtccaggat ctccaggacc tggctctaca acctcgataa gaacctcgac 1440
aacgaataca tctccaccct caactacctc tacgacagga tcaccaatga gctgaccaac 1500
tccttctcca agaactccgc cgccaacgtg aactatattg ccgaaactct gggaatcaac 1560
cctgccgaat tcgccgaaca atatttcaga ttcagcatta tgaaagagca gaaaaacctc 1620
ggattcaata tcaccaagct cagggaagtg atgctggaca ggaaggatat gtccgagatc 1680
aggaaaaatc ataaggtgtt cgactccatc aggaccaagg tctacaccat gatggacttt 1740
gtgatttata ggtattacat cgaagaggat gccaaggtgg ctgccgccaa taagtccctc 1800
cccgataatg agaagtccct gagcgagaag gatatctttg tgattaacct gaggggctcc 1860
ttcaacgacg accagaagga tgccctctac tacgatgaag ctaatagaat ttggagaaag 1920
ctcgaaaata tcatgcacaa catcaaggaa tttaggggaa acaagacaag agagtataag 1980
aagaaggacg cccctagact gcccagaatc ctgcccgctg gccgtgatgt ttccgccttc 2040
agcaaactca tgtatgccct gaccatgttc ctggatggca aggagatcaa cgacctcctg 2100
accaccctga ttaataaatt cgataacatc cagagcttcc tgaaggtgat gcctctcatc 2160
ggagtcaacg ctaagttcgt ggaggaatac gcctttttca aagactccgc caagatcgcc 2220
gatgagctga ggctgatcaa gtccttcgct agaatgggag aacctattgc cgatgccagg 2280
agggccatgt atatcgacgc catccgtatt ttaggaacca acctgtccta tgatgagctc 2340
aaggccctcg ccgacacctt ttccctggac gagaacggaa acaagctcaa gaaaggcaag 2400
cacggcatga gaaatttcat tattaataac gtgatcagca ataaaaggtt ccactacctg 2460
atcagatacg gtgatcctgc ccacctccat gagatcgcca aaaacgaggc cgtggtgaag 2520
ttcgtgctcg gcaggatcgc tgacatccag aaaaaacagg gccagaacgg caagaaccag 2580
atcgacaggt actacgaaac ttgtatcgga aaggataagg gcaagagcgt gagcgaaaag 2640
gtggacgctc tcacaaagat catcaccgga atgaactacg accaattcga caagaaaagg 2700
agcgtcattg aggacaccgg cagggaaaac gccgagaggg agaagtttaa aaagatcatc 2760
agcctgtacc tcaccgtgat ctaccacatc ctcaagaata ttgtcaatat caacgccagg 2820
tacgtcatcg gattccattg cgtcgagcgt gatgctcaac tgtacaagga gaaaggctac 2880
gacatcaatc tcaagaaact ggaagagaag ggattcagct ccgtcaccaa gctctgcgct 2940
ggcattgatg aaactgcccc cgataagaga aaggacgtgg aaaaggagat ggctgaaaga 3000
gccaaggaga gcattgacag cctcgagagc gccaacccca agctgtatgc caattacatc 3060
aaatacagcg acgagaagaa agccgaggag ttcaccaggc agattaacag ggagaaggcc 3120
aaaaccgccc tgaacgccta cctgaggaac accaagtgga atgtgatcat cagggaggac 3180
ctcctgagaa ttgacaacaa gacatgtacc ctgttcagaa acaaggccgt ccacctggaa 3240
gtggccaggt atgtccacgc ctatatcaac gacattgccg aggtcaattc ctacttccaa 3300
ctgtaccatt acatcatgca gagaattatc atgaatgaga ggtacgagaa aagcagcgga 3360
aaggtgtccg agtacttcga cgctgtgaat gacgagaaga agtacaacga taggctcctg 3420
aaactgctgt gtgtgccttt cggctactgt atccccaggt ttaagaacct gagcatcgag 3480
gccctgttcg ataggaacga ggccgccaag ttcgacaagg agaaaaagaa ggtgtccggc 3540
aattccggat ccgactacaa agaccatgac ggtgattata aagatcatga catcgattac 3600
aaggatgacg atgacaagta ataagctagc ataatcaacc tctggattac aaaatttgtg 3660
aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 3720
taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 3780
aatcctggtt agttcttgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga 3840
caggggctcg gctgttgggc actgacaatt ccgtggtgtt tatttgtgaa atttgtgatg 3900
ctattgcttt atttgtaacc atctagcttt atttgtgaaa tttgtgatgc tattgcttta 3960
tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 4020
tttcaggttc agggggagat gtgggaggtt ttttaaagcg gccgcaggaa cccctagtga 4080
tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg 4140
tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc 4200
tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 4260
tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 4320
ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 4380
cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 4440
ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg 4500
tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 4560
gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 4620
gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 4680
gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg 4740
gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc 4800
aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 4860
tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 4920
gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 4980
ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 5040
tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 5100
ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 5160
ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 5220
tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 5280
gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 5340
gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 5400
acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 5460
tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 5520
caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 5580
gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 5640
cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 5700
tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 5760
agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 5820
tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 5880
ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 5940
acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 6000
ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 6060
gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 6120
gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 6180
ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 6240
gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 6300
ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 6360
cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 6420
cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 6480
ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 6540
tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 6600
cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 6660
ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 6720
aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 6780
ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc 6840
cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag 6900
agggagtggc caactccatc actaggggtt cct 6933
<210> 9
<211> 6999
<212> DNA
<213> gRNA-NC Vector，pCasRxN-A1
<400> 9
gcggccattc ggtacaattc acgcgtgagg gcctatttcc catgattcct tcatatttgc 60
atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga 120
tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa 180
aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct 240
tggctttata tatcttgtgg aaaggacgaa acaccgaacc cctaccaact ggtcggggtt 300
tgaaactcac cagaagcgta ccatactctt tttttgaatt cgtattagtc atcgctatta 360
ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 420
gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 480
gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 540
tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 600
gccatccacg ctgttttgac ctccatagaa gattctagag ccaccatgag ccccaagaag 660
aagagaaagg tggaggccag catcgaaaaa aaaaagtcct tcgccaaggg catgggcgtg 720
aagtccacac tcgtgtccgg ctccaaagtg tacatgacaa ccttcgccga aggcagcgac 780
gccaggctgg aaaagatcgt ggagggcgac agcatcagga gcgtgaatga gggcgaggcc 840
ttcagcgctg aaatggccga taaaaacgcc ggctataaga tcggcaacgc caaattcagc 900
catcctaagg gctacgccgt ggtggctaac aaccctctgt atacaggacc cgtccagcag 960
gatatgctcg gcctgaagga aactctggaa aagaggtact tcggcgagag cgctgatggc 1020
aatgacaata tttgtatcca ggtgatccat aacatcctgg acattgaaaa aatcctcgcc 1080
gaatacatta ccaacgccgc ctacgccgtc aacaatatct ccggcctgga taaggacatt 1140
attggattcg gcaagttctc cacagtgtat acctacgacg aattcaaaga ccccgagcac 1200
catagggccg ctttcaacaa taacgataag ctcatcaacg ccatcaaggc ccagtatgac 1260
gagttcgaca acttcctcga taaccccaga ctcggctatt tcggccaggc ctttttcagc 1320
aaggagggca gaaattacat catcaattac ggcaacgaat gctatgacat tctggccctc 1380
ctgagcggac tgaggcactg ggtggtccat aacaacgaag aagagtccag gatctccagg 1440
acctggctct acaacctcga taagaacctc gacaacgaat acatctccac cctcaactac 1500
ctctacgaca ggatcaccaa tgagctgacc aactccttct ccaagaactc cgccgccaac 1560
gtgaactata ttgccgaaac tctgggaatc aaccctgccg aattcgccga acaatatttc 1620
agattcagca ttatgaaaga gcagaaaaac ctcggattca atatcaccaa gctcagggaa 1680
gtgatgctgg acaggaagga tatgtccgag atcaggaaaa atcataaggt gttcgactcc 1740
atcaggacca aggtctacac catgatggac tttgtgattt ataggtatta catcgaagag 1800
gatgccaagg tggctgccgc caataagtcc ctccccgata atgagaagtc cctgagcgag 1860
aaggatatct ttgtgattaa cctgaggggc tccttcaacg acgaccagaa ggatgccctc 1920
tactacgatg aagctaatag aatttggaga aagctcgaaa atatcatgca caacatcaag 1980
gaatttaggg gaaacaagac aagagagtat aagaagaagg acgcccctag actgcccaga 2040
atcctgcccg ctggccgtga tgtttccgcc ttcagcaaac tcatgtatgc cctgaccatg 2100
ttcctggatg gcaaggagat caacgacctc ctgaccaccc tgattaataa attcgataac 2160
atccagagct tcctgaaggt gatgcctctc atcggagtca acgctaagtt cgtggaggaa 2220
tacgcctttt tcaaagactc cgccaagatc gccgatgagc tgaggctgat caagtccttc 2280
gctagaatgg gagaacctat tgccgatgcc aggagggcca tgtatatcga cgccatccgt 2340
attttaggaa ccaacctgtc ctatgatgag ctcaaggccc tcgccgacac cttttccctg 2400
gacgagaacg gaaacaagct caagaaaggc aagcacggca tgagaaattt cattattaat 2460
aacgtgatca gcaataaaag gttccactac ctgatcagat acggtgatcc tgcccacctc 2520
catgagatcg ccaaaaacga ggccgtggtg aagttcgtgc tcggcaggat cgctgacatc 2580
cagaaaaaac agggccagaa cggcaagaac cagatcgaca ggtactacga aacttgtatc 2640
ggaaaggata agggcaagag cgtgagcgaa aaggtggacg ctctcacaaa gatcatcacc 2700
ggaatgaact acgaccaatt cgacaagaaa aggagcgtca ttgaggacac cggcagggaa 2760
aacgccgaga gggagaagtt taaaaagatc atcagcctgt acctcaccgt gatctaccac 2820
atcctcaaga atattgtcaa tatcaacgcc aggtacgtca tcggattcca ttgcgtcgag 2880
cgtgatgctc aactgtacaa ggagaaaggc tacgacatca atctcaagaa actggaagag 2940
aagggattca gctccgtcac caagctctgc gctggcattg atgaaactgc ccccgataag 3000
agaaaggacg tggaaaagga gatggctgaa agagccaagg agagcattga cagcctcgag 3060
agcgccaacc ccaagctgta tgccaattac atcaaataca gcgacgagaa gaaagccgag 3120
gagttcacca ggcagattaa cagggagaag gccaaaaccg ccctgaacgc ctacctgagg 3180
aacaccaagt ggaatgtgat catcagggag gacctcctga gaattgacaa caagacatgt 3240
accctgttca gaaacaaggc cgtccacctg gaagtggcca ggtatgtcca cgcctatatc 3300
aacgacattg ccgaggtcaa ttcctacttc caactgtacc attacatcat gcagagaatt 3360
atcatgaatg agaggtacga gaaaagcagc ggaaaggtgt ccgagtactt cgacgctgtg 3420
aatgacgaga agaagtacaa cgataggctc ctgaaactgc tgtgtgtgcc tttcggctac 3480
tgtatcccca ggtttaagaa cctgagcatc gaggccctgt tcgataggaa cgaggccgcc 3540
aagttcgaca aggagaaaaa gaaggtgtcc ggcaattccg gatccggacc taagaaaaag 3600
aggaaggtgg cggcggctga ctacaaagac catgacggtg attataaaga tcatgacatc 3660
gattacaagg atgacgatga caagtaataa gctagcataa tcaacctctg gattacaaaa 3720
tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 3780
ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 3840
tgtataaatc ctggttagtt cttgccacgg cggaactcat cgccgcctgc cttgcccgct 3900
gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgtttatt tgtgaaattt 3960
gtgatgctat tgctttattt gtaaccatct agctttattt gtgaaatttg tgatgctatt 4020
gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat 4080
tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcggccg caggaacccc 4140
tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 4200
caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 4260
gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 4320
accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg 4380
tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 4440
cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg 4500
ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga 4560
tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac 4620
gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 4680
tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 4740
aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat 4800
tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 4860
cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 4920
acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 4980
acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 5040
aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 5100
tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 5160
gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 5220
tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 5280
aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 5340
cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 5400
agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 5460
ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 5520
tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 5580
tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 5640
caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 5700
accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 5760
attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 5820
ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 5880
taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 5940
taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 6000
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 6060
agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 6120
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 6180
ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 6240
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 6300
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 6360
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 6420
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 6480
tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 6540
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 6600
acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 6660
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 6720
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 6780
ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 6840
ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc gctcgctcac 6900
tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc 6960
gcagagaggg agtggccaac tccatcacta ggggttcct 6999
<210> 10
<211> 939
<212> DNA
<213> GW1-seq
<400> 10
gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca 60
ttcgattagt gaacggatct cgacggttaa cttttaaaag aaaagggggg attggggggt 120
acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac 180
aaaaacaaat tacaaaaatt caaaatttta tcgatactag tattatgccc agtacatgac 240
cttatgggac tttcctactt ggcagtacat ctacgctagc gagggcctat ttcccatgat 300
tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 360
tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 420
gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 480
gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg aacccctacc 540
aactggtcgg ggtttgaaac gggtcttcga gaagaccttt tttttgaatt cgtattagtc 600
atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 660
gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 720
caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 780
ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc 840
gcctggagac gccatccacg ctgttttgac ctccatagaa gattctagag ctagcgaatt 900
cgaatttaaa tcggatccgc ggccgcaagg atctgcgat 939
<210> 11
<211> 26
<212> DNA
<213> 13d-GW1-UnF
<400> 11
agtgaacgga tctcgacggt taactt 26
<210> 12
<211> 22
<212> DNA
<213> 13d-GW1-D1-R
<400> 12
gtagatgtac tgccaagtag ga 22
<210> 13
<211> 50
<212> DNA
<213> 13d-GW1-D2-F
<400> 13
cctacttggc agtacatcta cgctagcgag ggcctatttc ccatgattcc 50
<210> 14
<211> 48
<212> DNA
<213> 13d-GW1-D2-inR
<400> 14
caaaccccga ccagttggta ggggttcggt gtttcgtcct ttccacaa 48
<210> 15
<211> 48
<212> DNA
<213> 13d-GW1-D2-R
<400> 15
aaggtcttct cgaagacccg tttcaaaccc cgaccagttg gtaggggt 48
<210> 16
<211> 57
<212> DNA
<213> 13d-GW1-D3-F
<400> 16
gaaacgggtc ttcgagaaga cctttttttt gaattcgtat tagtcatcgc tattacc 57
<210> 17
<211> 32
<212> DNA
<213> 13d-GW1-UnR
<400> 17
gatttaaatt cgaattcgct agctctagaa tc 32
<210> 18
<211> 1802
<212> DNA
<213> GW2-seq
<400> 18
ggatccttcg aagcccctct ccctcccccc cccctaacgt tactggccga agccgcttgg 60
aataaggccg gtgtgcgttt gtctatatgt gattttccac catattgccg tcttttggca 120
atgtgagggc ccggaaacct ggccctgtct ttttgacgag cattcctagg ggtctttccc 180
ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag 240
cttcttgagg acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg 300
gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac 360
aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa 420
gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc 480
tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc 540
ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataagct tgccaatgac 600
cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac 660
cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca 720
catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg 780
caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt 840
cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg 900
gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc 960
gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc 1020
cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac 1080
ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt 1140
cgagtgcccg aaggaccgcg cgacctggtg catgacccgc aagcccggtg cctgataagc 1200
ggccgcaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 1260
gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 1320
tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 1380
gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 1440
cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 1500
ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 1560
cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg 1620
ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 1680
gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 1740
cgtctccgcc ttcgccctca gacgagtcgg atctcccttt ggccgcctcc ccgcctggta 1800
cc 1802
<210> 19
<211> 59
<212> DNA
<213> 13d-GW2-UnF
<400> 19
agcgaattcg aatttaaatc ggatccttcg aagcccctct ccctcccccc cccctaacg 59
<210> 20
<211> 39
<212> DNA
<213> 13d-GW2-D1-R
<400> 20
ttatcatcgt gtttttcaaa ggaaaaccac gtccccgtg 39
<210> 21
<211> 55
<212> DNA
<213> 13d-GW2-D2-F
<400> 21
ttcctttgaa aaacacgatg ataagcttgc caatgaccga gtacaagccc acggt 55
<210> 22
<211> 42
<212> DNA
<213> 13d-GW2-D2-R
<400> 22
ggttgattgc ggccgcttat caggcaccgg gcttgcgggt ca 42
<210> 23
<211> 41
<212> DNA
<213> 13d-GW2-D3-F
<400> 23
tgataagcgg ccgcaatcaa cctctggatt acaaaatttg t 41
<210> 24
<211> 31
<212> DNA
<213> 13d-GW2-UnR
<400> 24
cttgtaagtc attggtctta aaggtaccag g 31
<210> 25
<211> 3781
<212> DNA
<213> 13d-CasRx
<400> 25
tctagagcca ccatgatcga aaaaaaaaag tccttcgcca agggcatggg cgtgaagtcc 60
acactcgtgt ccggctccaa agtgtacatg acaaccttcg ccgaaggcag cgacgccagg 120
ctggaaaaga tcgtggaggg cgacagcatc aggagcgtga atgagggcga ggccttcagc 180
gctgaaatgg ccgataaaaa cgccggctat aagatcggca acgccaaatt cagccatcct 240
aagggctacg ccgtggtggc taacaaccct ctgtatacag gacccgtcca gcaggatatg 300
ctcggcctga aggaaactct ggaaaagagg tacttcggcg agagcgctga tggcaatgac 360
aatatttgta tccaggtgat ccataacatc ctggacattg aaaaaatcct cgccgaatac 420
attaccaacg ccgcctacgc cgtcaacaat atctccggcc tggataagga cattattgga 480
ttcggcaagt tctccacagt gtatacctac gacgaattca aagaccccga gcaccatagg 540
gccgctttca acaataacga taagctcatc aacgccatca aggcccagta tgacgagttc 600
gacaacttcc tcgataaccc cagactcggc tatttcggcc aggccttttt cagcaaggag 660
ggcagaaatt acatcatcaa ttacggcaac gaatgctatg acattctggc cctcctgagc 720
ggactgaggc actgggtggt ccataacaac gaagaagagt ccaggatctc caggacctgg 780
ctctacaacc tcgataagaa cctcgacaac gaatacatct ccaccctcaa ctacctctac 840
gacaggatca ccaatgagct gaccaactcc ttctccaaga actccgccgc caacgtgaac 900
tatattgccg aaactctggg aatcaaccct gccgaattcg ccgaacaata tttcagattc 960
agcattatga aagagcagaa aaacctcgga ttcaatatca ccaagctcag ggaagtgatg 1020
ctggacagga aggatatgtc cgagatcagg aaaaatcata aggtgttcga ctccatcagg 1080
accaaggtct acaccatgat ggactttgtg atttataggt attacatcga agaggatgcc 1140
aaggtggctg ccgccaataa gtccctcccc gataatgaga agtccctgag cgagaaggat 1200
atctttgtga ttaacctgag gggctccttc aacgacgacc agaaggatgc cctctactac 1260
gatgaagcta atagaatttg gagaaagctc gaaaatatca tgcacaacat caaggaattt 1320
aggggaaaca agacaagaga gtataagaag aaggacgccc ctagactgcc cagaatcctg 1380
cccgctggcc gtgatgtttc cgccttcagc aaactcatgt atgccctgac catgttcctg 1440
gatggcaagg agatcaacga cctcctgacc accctgatta ataaattcga taacatccag 1500
agcttcctga aggtgatgcc tctcatcgga gtcaacgcta agttcgtgga ggaatacgcc 1560
tttttcaaag actccgccaa gatcgccgat gagctgaggc tgatcaagtc cttcgctaga 1620
atgggagaac ctattgccga tgccaggagg gccatgtata tcgacgccat ccgtatttta 1680
ggaaccaacc tgtcctatga tgagctcaag gccctcgccg acaccttttc cctggacgag 1740
aacggaaaca agctcaagaa aggcaagcac ggcatgagaa atttcattat taataacgtg 1800
atcagcaata aaaggttcca ctacctgatc agatacggtg atcctgccca cctccatgag 1860
atcgccaaaa acgaggccgt ggtgaagttc gtgctcggca ggatcgctga catccagaaa 1920
aaacagggcc agaacggcaa gaaccagatc gacaggtact acgaaacttg tatcggaaag 1980
gataagggca agagcgtgag cgaaaaggtg gacgctctca caaagatcat caccggaatg 2040
aactacgacc aattcgacaa gaaaaggagc gtcattgagg acaccggcag ggaaaacgcc 2100
gagagggaga agtttaaaaa gatcatcagc ctgtacctca ccgtgatcta ccacatcctc 2160
aagaatattg tcaatatcaa cgccaggtac gtcatcggat tccattgcgt cgagcgtgat 2220
gctcaactgt acaaggagaa aggctacgac atcaatctca agaaactgga agagaaggga 2280
ttcagctccg tcaccaagct ctgcgctggc attgatgaaa ctgcccccga taagagaaag 2340
gacgtggaaa aggagatggc tgaaagagcc aaggagagca ttgacagcct cgagagcgcc 2400
aaccccaagc tgtatgccaa ttacatcaaa tacagcgacg agaagaaagc cgaggagttc 2460
accaggcaga ttaacaggga gaaggccaaa accgccctga acgcctacct gaggaacacc 2520
aagtggaatg tgatcatcag ggaggacctc ctgagaattg acaacaagac atgtaccctg 2580
ttcagaaaca aggccgtcca cctggaagtg gccaggtatg tccacgccta tatcaacgac 2640
attgccgagg tcaattccta cttccaactg taccattaca tcatgcagag aattatcatg 2700
aatgagaggt acgagaaaag cagcggaaag gtgtccgagt acttcgacgc tgtgaatgac 2760
gagaagaagt acaacgatag gctcctgaaa ctgctgtgtg tgcctttcgg ctactgtatc 2820
cccaggttta agaacctgag catcgaggcc ctgttcgata ggaacgaggc cgccaagttc 2880
gacaaggaga aaaagaaggt gtccggcaat tccggatccg actacaaaga ccatgacggt 2940
gattataaag atcatgacat cgattacaag gatgacgatg acaagggtag cggcagtgga 3000
gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg cccagtgagc 3060
aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 3120
aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 3180
accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 3240
accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 3300
ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 3360
gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 3420
atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 3480
tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 3540
gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 3600
cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 3660
acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 3720
ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta attcgaagcc 3780
c 3781
<210> 26
<211> 59
<212> DNA
<213> 13d-GW3-D1-UnF
<400> 26
tgttttgacc tccatagaag attctagagc caccatgatc gaaaaaaaaa agtccttcg 59
<210> 27
<211> 59
<212> DNA
<213> 13d-GW3-D1-R
<400> 27
atgatcttta taatcaccgt catggtcttt gtagtcggat ccggaattgc cggacacct 59
<210> 28
<211> 49
<212> DNA
<213> 13d-GW3-D2-F
<400> 28
aagaccatga cggtgattat aaagatcatg acatcgatta caaggatga 49
<210> 29
<211> 59
<212> DNA
<213> 13d-GW3-D2-F-in
<400> 29
gatcatgaca tcgattacaa ggatgacgat gacaagggta gcggcagtgg agagggcag 59
<210> 30
<211> 59
<212> DNA
<213> 13d-GW3-D2-UnR
<400> 30
taacgttagg ggggggggag ggagaggggc ttcgaattac ttgtacagct cgtccatgc 59
<210> 31
<211> 22
<212> DNA
<213> non-targeting gRNA
<400> 31
tcaccagaag cgtaccatac tc 22
<210> 32
<211> 26
<212> DNA
<213> gRNA-NT-F
<400> 32
aaactcacca gaagcgtacc atactc 26
<210> 33
<211> 26
<212> DNA
<213> gRNA-NT-R
<400> 33
aaaagagtat ggtacgcttc tggtga 26
<210> 34
<211> 26
<212> DNA
<213> hsa_circ_0001821-gRNA1-F
<400> 34
aaacaaagat caggcctcaa gcccag 26
<210> 35
<211> 26
<212> DNA
<213> hsa_circ_0001821-gRNA1-R
<400> 35
aaaactgggc ttgaggcctg atcttt 26
<210> 36
<211> 29
<212> DNA
<213> hsa_circ_0001821-gRNA2-F
<400> 36
aaaccaaaag atcaggcctc aagcccagc 29
<210> 37
<211> 29
<212> DNA
<213> hsa_circ_0001821-gRNA2-R
<400> 37
aaaagctggg cttgaggcct gatcttttg 29
<210> 38
<211> 32
<212> DNA
<213> hsa_circ_0001821-gRNA3-F
<400> 38
aaacccaaaa gatcaggcct caagcccagc tg 32
<210> 39
<211> 32
<212> DNA
<213> hsa_circ_0001821-gRNA3-R
<400> 39
aaaacagctg ggcttgaggc ctgatctttt gg 32
Claims (3)
1.一种试剂盒在快速构建环形RNA敲低载体中的应用，其特征在于，包含LinearizedCasRx Vector、DNA Ligase Mix、10×Ligase Buffer、Nuclease-free Water、10μM的U6Primer、gRNA-NC Vector共6种成分；所述Linearized CasRx Vector选自LinearizedCasRx Vector，From pCasRx-L1；Linearized CasRx Vector，From pCasRxN-L1；Linearized CasRx Vector，From pCasRx-A1和Linearized CasRx Vector，From pCasRxN-A1中的一个或多个；所述Linearized CasRx Vector的序列组成为TTTT-U6 terminator-CMV promoter-CasRx-Flag-EGFP-IRES-PuroR- WPRE-backbone-U6 promoter-CasRxDR30-AAAC；所述Linearized CasRx Vector，From pCasRx-L1和 Linearized CasRxVector，From pCasRxN-L1为慢病毒载体骨架；所述Linearized CasRx Vector，FrompCasRx-A1和Linearized CasRx Vector，From pCasRxN-A1为ssAAV载体骨架；所述Linearized CasRx Vector，From pCasRxN-L1和 Linearized CasRx Vector，FrompCasRxN-A1中CasRx的5’和3’末端分别添加有核定位信号NLS序列；所述Linearized CasRxVector，From pCasRx-L1的核苷酸序列如SEQ ID NO.1；所述Linearized CasRx Vector，From pCasRxN-L1的核苷酸序列如SEQ ID NO.2；所述Linearized CasRx Vector，FrompCasRx-A1的核苷酸序列如SEQ ID NO.3；所述Linearized CasRx Vector，From pCasRxN-A1的核苷酸序列如SEQ ID NO.4；所述Linearized CasRx Vector，From pCasRx-L1为pCasRx-L0经BpiI双酶切线性化后回收制备而成；所述Linearized CasRx Vector，FrompCasRxN-L1为pCasRxN-L0经BpiI双酶切线性化后回收制备而成；所述Linearized CasRxVector，From pCasRx-A1为pCasRx-A0经BpiI双酶切线性化后回收制备而成；所述Linearized CasRx Vector，From pCasRxN-A1为pCasRxN-A0经BpiI双酶切线性化后回收制备而成；所述gRNA-NC Vector选自gRNA-NC Vector，pCasRx-L1、gRNA-NC Vector，pCasRxN-L1、gRNA-NC Vector，pCasRx-A1和gRNA-NC Vector，pCasRxN-A1中的一个或多个；所述Linearized CasRx Vector的浓度为10ng/μL；所述试剂盒的连接反应为5μL体系，且gRNA(50μM)退火双链产物与Linearized CasRx Vector的比例为1：2（V/V）；所述DNA LigaseMix包含500U/μL的T4 DNA ligase、pH为7.5，20mM的Tris-HCl、50mM KCl、1mM DTT、0.1mMEDTA和50%甘油；所述10×Ligase Buffer包含pH为7.8，400mM Tris-HCl、100mM MgCl2、100mM DTT、5mM ATP和25% PEG 4000；所述10μM的U6 Primer为合成的U6引物，核苷酸序列信息如SEQ ID NO.5所示，并经PAGE纯化，使用时Nuclease-free Water稀释；所述gRNA-NCVector的序列组成为backbone-U6 promoter-CasRx DR30-non-targeting gRNA-U6terminator-CMV promoter-CasRx-Flag-EGFP-IRES-PuroR-WPRE；所述gRNA-NC Vector，pCasRx- L1和gRNA-NC Vector，pCasRxN-L1为慢病毒载体骨架；所述gRNA-NC Vector，pCasRx-A1和gRNA-NC Vector，pCasRxN-A1为ssAAV载体骨架；所述gRNA-NC Vector,pCasRxN-L1和gRNA-NC Vector，pCasRxN-A1中CasRx的5’和3’末端分别添加有核定位信号NLS序列；所述gRNA-NC Vector，pCasRx-L1的核苷酸序列如SEQ ID NO.6；所述gRNA-NCVector，pCasRxN-L1的核苷酸序列如SEQ ID NO.7；所述gRNA-NC Vector，pCasRx-A1的核苷酸序列如SEQ ID NO.8；所述gRNA-NC Vector，pCasRxN-A1的核苷酸序列如SEQ ID NO.9。
2.一种利用权利要求1所述试剂盒在构建目标环形RNA的CasRx/gRNA整合型表达载体中的应用。
3.一种CasRx/gRNA整合型表达载体在制备可持续敲低目标环形RNA的稳定细胞系、动物体或组织脏器模型中的应用，所述CasRx/gRNA整合型表达载体采用权利要求1所述试剂盒制成。
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| WO2020047489A1 (en) * | 2018-08-31 | 2020-03-05 | The Regents Of The University Of California | Directed pseudouridylation of rna |
| CN112143701A (zh) * | 2019-06-26 | 2020-12-29 | 中国科学院脑科学与智能技术卓越创新中心 | 基于rna定点编辑的抑制脉络膜新生血管形成的方法及试剂 |
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| WO2020047489A1 (en) * | 2018-08-31 | 2020-03-05 | The Regents Of The University Of California | Directed pseudouridylation of rna |
| CN110257420A (zh) * | 2019-06-14 | 2019-09-20 | 中国科学院武汉植物园 | 基于CasRx的植物基因沉默载体及其构建方法和应用 |
| CN112143701A (zh) * | 2019-06-26 | 2020-12-29 | 中国科学院脑科学与智能技术卓越创新中心 | 基于rna定点编辑的抑制脉络膜新生血管形成的方法及试剂 |
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