CN115029374B - 一种用于骨干载体的pegRNA表达框及相应骨干载体和应用 - Google Patents

一种用于骨干载体的pegRNA表达框及相应骨干载体和应用 Download PDF

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CN115029374B
CN115029374B CN202210729325.8A CN202210729325A CN115029374B CN 115029374 B CN115029374 B CN 115029374B CN 202210729325 A CN202210729325 A CN 202210729325A CN 115029374 B CN115029374 B CN 115029374B
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李娟�
许蓉芳
秦瑞英
魏鹏程
金珊
刘小双
陈俐克
丁健
李亦臻
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Hefei Jiangu Biotechnology Co ltd
Rice Research Institute of Anhui Academy of Agricultural Sciences
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Abstract

本发明公开了一种用于骨干载体的pegRNA表达框及相应骨干载体和应用。本发明的骨干载体包括融合蛋白、pegRNA；所述融合蛋白为Cas9切刻酶或其变体、反转录酶组成的融合蛋白。pegRNA包含靶向目的DNA片段的sgRNA、逆转录模板和引物结合位点、连接得到的RNA分子。本发明所述骨干质粒载体中，pegRNA表达框和融合蛋白表达框位于同一双元载体中，pegRNA表达框依次由35S‑CmYLCV‑U6复合启动子、tRNA基因序列、壮观霉素抗性基因SpR、RNA核酶HDV序列、EQ序列和polyT‑HSPt复合终止子组成；Cas9核酸酶表达框由ZmUBI启动子，融合蛋白编码序列和35s终止子依次构成。本发明的enpPE2引导编辑系统不仅大大提高了编辑靶点的编辑效率，并能获得纯合突变植株，具有很好的应用前景。

Description

一种用于骨干载体的pegRNA表达框及相应骨干载体和应用
技术领域
本发明属于生物技术领域，具体涉及引导编辑系统及其在基因组碱基编辑中的应用。
背景技术
基因编辑技术可有目的的实现基因组中特定DNA片段的敲除、插入和替换。CRISPR/Cas以其易用性和高效性的优点成为主流基因组编辑技术，正日益深刻的影响着植物学的发展。目前多数相关研究利用CRISPR-Cas系统在植物基因组特定位点产生DNA双链断裂(Double strand break,DSB)，并通过错误倾向性的非同源末端连接(Non-homologousend joining,NHEJ)修复失活目标基因。由于 NHEJ修复介导的碱基插入缺失存在一定的随机性，难以实现精确的基因组编辑。借助于同源介导修复(homology-directed repair,HDR)机制，CRISPR-Cas系统可在外源DNA供体(donor)的指导下实现精准的碱基替换或片段插入缺失。但是在植物细胞中，由于重组频率偏低和DNAdonor的递送困难，CRISPR介导的HDR 效率显著受限，往往难以高效的实现基因组精确编辑。
2019年底，David Liu研究组报道了不同于单碱基编辑的基因组精确编辑技术，即引导编辑(prime editing,PE)系统。该系统利用nSpCas9(H840A)和工程化改造M-MLV RT反转录酶(Moloney murine leukemia virus reverse transcriptase) 融合构建引导编辑器(prime editor)，并利用prime editing guide RNA(pegRNA)最终实现靶位点的基因编辑。pegRNA由3个部分组成，包括single-guide RNA (sgRNA)、引物结合位点(PrimeBinding Site，PBS)和储存有靶向位点编辑信息的反转录模板(RT template)。由pegRNA中guide RNA部分引导在人细胞基因组靶位置附近形成编辑链上的单链切口，进而通过pegRNA中的PBS序列引导以含有目标编辑序列的逆转录模板将突变精确导入基因组中。
相对于其他的编辑技术，引导编辑展示了优越性：1、引导编辑可完成的编辑类型广泛，不仅可以实现12种任意类型的碱基替换，还可精确高效的导入小片段插入以及80bp以内的片段缺失。2、引导编辑受PAM的限制相对较小，在长达33nt的PAM远端序列内，多个位点都可以高效编辑。3、引导编辑更为精准，当在目标位点附近有多个相同碱基时，不会受到碱基编辑器bystander mutation问题的困扰。
在植物中，国内多个课题组均在水稻、玉米等建立了植物引导编辑系统 (PPE)，可灵活地在作物中实现单碱基和多碱基替换、小片段插入缺失等其他编辑工具无法完成的多种精确突变，极大拓展了植物基因组精准编辑系统。但目前植物引导编辑效率偏低，通常在8％以下，所产生的突变体多为嵌合突变，严重制约了其应用。因此，有必要开发出一种高效的植物引导编辑系统，更好的为植物基因功能解析和作物遗传改良提供有利工具。
发明内容
本发明所要解决的技术问题是如何提高植物引导编辑系统的精确编辑效率。
为解决上述技术问题，本发明首先提供了一种用于骨干载体的pegRNA表达框，其特征在于，所述pegRNA表达框包含启动子、tRNA基因序列、壮观霉素抗性基因SpR、EQ序列、RNA核酶HDV序列和终止子，其中，所述tRNA基因的核苷酸序列如Seq ID No.1第1274至1345位所示，所述壮观霉素抗性基因 SpR的核苷酸序列如Seq ID No.1第1452至2558位所示，所述EQ序列的核苷酸序列如Seq ID No.1第2566至2607位所示，RNA核酶HDV的核苷酸序列如Seq ID No.1第2608至2675位所示。
优选地，所述pegRNA表达框的核苷酸序列如序列表中Seq ID No.1的第274 至2954位所示，其中，所述启动子为35S-CmYLCV-U6复合启动子，所述终止子为polyT-HSPt复合终止子。
另一方面，本发明提供一种高效的植物引导编辑系统。所述植物引导编辑系统含有pegRNA，所述pegRNA为由靶向目的DNA片段的sgRNA、逆转录模板 (RT)和引物结合位点(PBS)连接得到的RNA分子。此外，在pegRNA的3’增加 EQ结构，避免pegRNA的3’被RNA酶降解，其次使用35S-CmYLCV-U6复合启动子驱动pegRNA的表达，以增强pegRNA的表达。
所述植物引导编辑系统还含有Cas9切口酶(H840A)或变体与逆转录酶 MMLV融合所形成的融合蛋白。通过使用Cas9切口酶变体(H840A/R221K/N394K) 以及不同类型的核定位信号NLS，产生优化的融合蛋白结构，提高其作用活性。
本发明还提供了一种骨干载体，所述载体含有pegRNA的表达框和融合蛋白表达框。所述向导RNA表达框的核苷酸序列如Seq ID No.1中第274至2954位所示，所述Cas9核酸酶表达框的核苷酸序列如Seq ID No.1中第2961至11653 位所示，其特征在于，
所述pegRNA表达框依次包括：35S-CmYLCV-U6复合启动子、tRNA基因序列、壮观霉素抗性基因SpR、EQ序列、RNA核酶HDV序列和polyT-HSPt复合终止子。其中，所述35S-CmYLCV-U6复合启动子的核苷酸序列如Seq ID No.1 第274至1266位所示，tRNA基因的核苷酸序列如Seq ID No.1第1274至1345 位所示，壮观霉素抗性基因SpR的核苷酸序列如SeqID No.1第1452至2558位所示，EQ序列的核苷酸序列如Seq ID No.1第2566至2607位所示，RNA核酶 HDV的核苷酸序列如Seq ID No.1第2608至2675位所示，polyT-HSPt复合终止子的核苷酸序列如Seq ID No.1第2676至2954位所示。
在本发明中，SpR基因两端分别存在反向排列的BsaI内切酶识别位点(剪切位点如Seq ID No.1第1346位和2559位所示)，用于插入目的基因sgRNA、sgRNA 骨架、相应的逆转录模板(RT)和引物结合位点(PBS)片段。
(2)所述融合蛋白表达框包括ZmUBI启动子、改造后的Cas9切刻酶或其变体编码序列、逆转录酶M-MLV编码序列和35s终止子，其中，其中，ZmUBI 启动子的核苷酸序列如SeqID No.1第2961至4939位所示，Cas9切刻酶的核苷酸序列如Seq ID No.1第4979至9079位所示，逆转录酶M-MLV RT的核苷酸序列如Seq ID No.1第9182至11254位所示，35s终止子的核苷酸序列如Seq ID No.1 第11389至11653位所示。此外，在Cas9切刻酶或其变体的5’端存在一个核定位信号SV40 NLS，其核苷酸序列如Seq ID No.1第4958至4978位所示；Cas9 切刻酶与逆转录酶M-MLV RT编码序列之间含有33aa的连接序列(linker)，其核苷酸序列如Seq ID No.1第9080至9181位所示；M-MLV RT编码序列的3’端含有核定位信号SV40 NLS和CY NLS(其核苷酸序列如Seq ID No.1第11258 至11314位所示)，本发明中采用了CY NLS，可以进一步帮助蛋白进入细胞核。
(3)所述骨干质粒载体还包括T-DNA的左、右边界序列，其中所述左边界的核苷酸序列如Seq ID No.1第14035至14060位所示，所述右边界的核苷酸序列如Seq ID No.1第1至26位所示；所述pegRNA表达框和所述融合蛋白表达框位于所述左边界和所述右边界之间。
(4)所述载体还可含有抗性标记基因。
本发明还提供了一种构建用于引导编辑的重组载体的方法，该方法包括：
按照目的基因的编码序列和突变类型，选择sgRNA序列，得到相应的逆转录模板(RT)和引物结合位点(PBS)序列。用BsaI内切酶切开本发明提供的骨干质粒载体，利用含BsaI的Golden Gate反应，将sgRNA序列、sgRNA骨架序列、RT和PBS序列、8bp linker序列替换壮观霉素抗性基因，形成用于作物目的基因的引导编辑重组载体。
另一方面，本发明提供一种宿主菌，其特征在于，所述宿主菌包含上述的重组载体。
本发明还提供了一种引导编辑系统在作物基因打靶中的应用，将所述重组载体应用于生物突变体获得，具体而言，将上面获得的重组载体转入植物细胞，比如，通过农杆菌介导法，使细胞同时含有针对靶标基因的pegRNA和融合蛋白；并对生物体的基因组进行编辑，获得生物突变体。
上述的应用或方法中，基因组序列的编辑包括基因组序列的碱基替换(如单碱基替换和多碱基替换)、碱基插入(如单碱基插入和多碱基插入)和碱基删除(如单碱基删除和多碱基删除)。在本发明的具体实施例中，所述基因组序列的编辑为基因组序列的单个碱基替换、插入或缺失。
本发明通过在pegRNA的3’增加EQ序列(如Seq ID No.1第2566至2607 位所示)，避免了pegRNA的3’被RNA酶降解，使用35S-CmYLCV-U6复合启动子提高pegRNA的表达，并且使用Cas9切口酶变体(H840A/R221K/N394K) 和优化NLS，产生优化的融合蛋白结构，构建了新型植物引导编辑系统enpPE2。利用本发明的引导编辑系统不仅可以有效地、大幅度地提高植物中先导编辑系统的编辑效率，还能获得纯合突变植株，具有很好的应用前景。
附图说明
图1为引导编辑系统骨干载体PHUC411-enpPE2的示意图；
图2为靶点pegRNA的结构示意图；
实施例
以下实施例便于更好地理解本发明，但并不限制本发明。
实施例1、引导编辑系统的设计
本实施例的引导编辑系统的骨干载体包括pegRNA表达盒、nCas9变体和 M-MLV RT组成的融合蛋白的表达盒。引导编辑系统表达载体的构建包括构建以上两部分。分别设计好这两个表达盒，并连入pCambia骨架载体中。上述两个表达盒是本实施例的引导编辑系统的表达载体的特有部分，其还可以包括一些常规载体所具有的一般结构，这里不再累述。
一、复合启动子驱动pegRNA表达的载体构建
复合启动子驱动pegRNA表达盒依次由35S-CmYLCV-U6复合启动子、tRNA 基因序列、壮观霉素抗性基因SpR、RNA核酶HDV序列、EQ序列和polyT-HSPt 复合终止子组成。pegRNA表达盒苏州金唯智生物科技有限公司合成，两端带上 HindIII酶切位点，连接于PUC57-AMP载体上，并装载入大肠杆菌XL-blue菌株中。利用酶切连接反应，将pegRNA表达盒连入pCambia骨架载体中。
二、融合蛋白的表达盒的载体构建
设计引物对R221K FP/RP、N394K FP/RP，根据全式金多点突试剂盒操作，将SpCas9(H840A)突变成SpCas9(H840A R22K N394K)。M-MLV RT序列基因是本实验室设计的，来自于pHUN411-PE2载体。该载体已由本实验室申请了国家发明专利(一种引导编辑系统介导作物产生内源除草剂抗性的方法，专利申请号：2021105065820)。
根据Gibson拼接原理，设计引物，分别扩增得到SpCas9(H840A R22K N394K) 序列、MLV序列(引物具体如表1所示)，并且在SpCas9(H840A R22K N394K)和MLV之间，及MLV的3’带有不同类型的核定位信号NLS，拼接后的融合序列成如下结构 (划横线部分代表NLS或linker序列)连接于PUC57-AMP 载体上，并且两端带有PstI和SacI酶切位点。利用酶切连接反应，将融合蛋白序列连入pCambia骨架载体中。
以上得到的最终载体序列如Seq ID No.1所示，并命名为PHUC411-enpPE2，结构示意图如图1所示。通过将上述骨干载体用BsaI酶切后，就可以将壮观霉素抗性基因SpR替换成sgRNA序列、sgRNA骨架序列、RT和PBS序列、8bp linker 序列，形成用于目的基因的引导编辑重组载体。
表1引物、引导编辑载体系统构建相关序列
三、目的基因的引导编辑载体构建
以水稻的OsPDS、OsALS、OsCDC48、OsACC为靶标基因，选择合适的靶标和突变类型，根据植物pegRNA设计网站PlantPegDesigner (http://www.plantgenomeediting.net/)的分析，得到相应靶标的SgRNA、RT和 PBS序列(如表2所示)。在每个靶点的PBS序列和EQ序列之间，根据pegRNA 的设计和优化工具pegLIT(https://peglit.liugroup.us/)的分析，添加8bp linker。因此，得到的每个靶标的pegRNA及pegRNA-EQ序列如表3所示。分别合成pegRNA及pegRNA-EQ的正向寡核苷酸链和可与之互补的反向寡核苷酸链，退火形成双链。
为了与已有的PE2系统进行效率比较，本实施例分别将四个靶标的pegRNA 连入pHUC411-enpPE和pHUN411-PE2。构建流程以OsPDS靶标为例，首先利用BsaI酶切切开pHUC411-enpPE和pHUN411-PE2载体，随后采用含BsaI的 GoldenGate反应体系，将pHUC411-enpPE和pHUN411-PE2载体和OsPDS的 pegRNA首尾相连，转入大肠杆菌中。通过选择具有卡那霉素抗性且不具壮观霉素抗性的菌斑，获得阳性转化子。经测序验证后，提取阳性质粒，构成用于植物 OsPDS基因的重组载体质粒，命名为pHUC411-enpPE-PDS和pHUN411-PE2-PDS载体。同理，得到pHUC411-enpPE-OsALS载体和pHUN411-PE2-OsALS载体、pHUC411-enpPE-OsCDC48载体和pHUN411-PE2-OsCDC48载体、 pHUC411-enpPE-OsACC载体和pHUN411-PE2-OsACC载体。利用冻融法将植物表达载体转入根癌农杆菌(Agrobacterium tumefaciens)EHA105中。
表2，编辑靶点的详细信息
表3，编辑靶点的pegRNA序列
四、水稻遗传转化及编辑效率检测
将上述转入了重组表达载体的根癌农杆菌进行农杆菌介导的遗传转化，该遗传转化、转化子筛选及转基因植株再生等参照Yongbo Duan(Yongbo Duan， Chenguang Zhai，etal.An efficient and high-throughput protocol for Agrobacterium mediatedtransformation based on phosphomannose isomerase positive selection inJaponica rice(Oryza sativa L.)[J].Plant Cell Report，2012.DOI 10.1007/s00299-012-1275-3.)等提出的方法。
每个载体共获得48株水稻T0苗。利用植物基因组小量提取试剂盒(天根生化公司)，提取转基因水稻植株的基因组DNA。以该DNA为模板，用Phusion 高保真DNA聚合酶(NEB公司)PCR扩增包含靶标区域的序列。
其中，对于OsALS靶点，采用引物对：5’-AACATTTGGGTATGGTGGTGCA-3’和5’-TTGCATAGAAGTACTTTATTCT-3’对OsALS进行PCR扩增，得到PCR扩增产物；对于OsACC靶点，采用引物对：5’-TTGATGACAGCCAAGGGAAATG-3’和5’-ATGCGGTCTGGGTTTATCTTGC-3’对OsACC进行PCR扩增，得到PCR 扩增产物；对于OsCDC48靶点，采用引物对： 5’-CGGAGGAAGGACAACCCTGAAG-3’和 5’-ATACAACGCAAATCTATCCATG-3’对OsCDC48进行PCR扩增，得到PCR 扩增产物；对于OsPDS靶点，采用引物对：5’-TCACACTGTTTTGTCGTCCACA’和5’-TTCCTGTTAAATGCACGCATGA-3’对OsPDS进行PCR扩增，得到PCR 扩增产物。将得到的PCR扩增产物进行Sanger测序及分析，测序结果只针对各pegRNA区进行分析。分别统计各靶点发生目标碱基替换的T0苗数，计算得出引导编辑器的编辑效率，结果见表4。
从表4中的编辑结果对比表明，对所有四个靶点获得的编辑植株中，已有的 PE2系统能够获得目标基因突变植株0～14株，编辑效率为0％～29.17％。而采用本发明的enpPE引导编辑系统后，每个靶点能够获得目标基因突变植株31～37 株，编辑效率为64.58％％～77.08％。而且，在OsALS、OsCDC48和OsPDS靶点，能够分别获得的16、19和10株纯合突变植株，远高于现有的PE2系统。因此，利用本发明的引导编辑系统不仅能够大大提高编辑效率，而且能够获得纯合突变植株，在作物品种改良中具有重要的应用价值。
表4，不同类型引导编辑器对水稻内源靶点的编辑效率汇总。
序列表
<120> 一种用于骨干载体的pegRNA表达框及相应骨干载体和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20294
<212> DNA
<213> pegRNA
<400> 1
taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta 60
aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc 120
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 180
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 240
cgacgttgta aaacgacggc cagtgccaag cttatggagt caaagattca aatagaggac 300
ctaacagaac tcgccgtaaa gactggcgaa cagttcatac agagtctctt acgactcaat 360
gacaagaaga aaatcttcgt caacatggtg gagcacgaca cacttgtcta ctccaaaaat 420
atcaaagata cagtctcaga agaccaaagg gcaattgaga cttttcaaca aagggtaata 480
tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt gaagatagtg 540
gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc catcgttgaa 600
gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa 660
aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgattg gcagacatac 720
tgtcccacaa atgaagatgg aatctgtaaa agaaaacgcg tgaaataatg cgtctgacaa 780
aggttaggtc ggctgccttt aatcaatacc aaagtggtcc ctaccacgat ggaaaaactg 840
tgcagtcggt ttggcttttt ctgacgaaca aataagattc gtggccgaca ggtgggggtc 900
caccatgtga aggcatcttc agactccaat aatggagcaa tgacgtaagg gcttacgaaa 960
taagtaaggg tagtttggga aatgtccact cacccgtcag tctataaata cttagcccct 1020
ccctcattgt taagggagca aaatctcaga gagatagtcc tagagagaga aagagagcaa 1080
gtagcctaga agtagtcaag gcggcgaagt attcaggcac gtggccagga agaagaaaag 1140
ccaagacgac gaaaacaggt aagagctaag catctagaaa gttgaaaaca atcttcaaaa 1200
gtcccacatc gcttagataa gaaaacgaag ctgagtttat atacagctag agtcgaagta 1260
gtgattgaac aaagcaccag tggtctagtg gtagaatagt accctgccac ggtacagacc 1320
cgggttcgat tcccggctgg tgcaagagac caacccagtg gacataagcc tgttcggttc 1380
gtaagctgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac 1440
gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgggg 1500
tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga 1560
tgttatggag cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaaaca 1620
tcatggggga agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca 1680
tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg 1740
gcggcctgaa gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg 1800
aaacaacgcg gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga 1860
gcgagattct ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc 1920
gttatccagc taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag 1980
gtatcttcga gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag 2040
aacatagcgt tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac 2100
aggatctatt tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg 2160
ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg 2220
gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt 2280
atcagcccgt catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg 2340
cctcgcgcgc agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg 2400
tagtcggcaa ataatgtcta gctagaaatt cgttcaagcc gacgccgctt cgcggcgcgg 2460
cttaactcaa gcgttagatg cactaagcac ataattgctc acagccaaac tatcaggtca 2520
agtctgcttt tattattttt aagcgtgcat aataagccgg tctcattgac gcggttctat 2580
ctagttacgc gttaaaccaa ctagaaaggc cggcatggtc ccagcctcct cgctggcgcc 2640
ggctgggcaa catgcttcgg catggcgaat gggacttttt tttgatatct ccggggctaa 2700
ttgaatatga agatgaagat gaaatatttg gtgtgtcaaa taaaaagctg gtgtgcttaa 2760
gtttgtgttt ttttcttggc ttgttgtgtt atgaatttgt ggctttttct aatattaaat 2820
gaatgtaaga tctcattata atgaataaac aaatgtttct ataatccatt gtgaatgttt 2880
tgttggatct cttctgcagc atataactac tgtatgtgct atggtatgga ctatggaata 2940
tgattaaaga taagaagctt tgcagcgtga cccggtcgtg cccctctcta gagataatga 3000
gcattgcatg tctaagttat aaaaaattac cacatatttt ttttgtcaca cttgtttgaa 3060
gtgcagttta tctatcttta tacatatatt taaactttac tctacgaata atataatcta 3120
tagtactaca ataatatcag tgttttagag aatcatataa atgaacagtt agacatggtc 3180
taaaggacaa ttgagtattt tgacaacagg actctacagt tttatctttt tagtgtgcat 3240
gtgttctcct ttttttttgc aaatagcttc acctatataa tacttcatcc attttattag 3300
tacatccatt tagggtttag ggttaatggt ttttatagac taattttttt agtacatcta 3360
ttttattcta ttttagcctc taaattaaga aaactaaaac tctattttag tttttttatt 3420
taataattta gatataaaat agaataaaat aaagtgacta aaaattaaac aaataccctt 3480
taagaaatta aaaaaactaa ggaaacattt ttcttgtttc gagtagataa tgccagcctg 3540
ttaaacgccg tcgacgagtc taacggacac caaccagcga accagcagcg tcgcgtcggg 3600
ccaagcgaag cagacggcac ggcatctctg tcgctgcctc tggacccctc tcgagagttc 3660
cgctccaccg ttggacttgc tccgctgtcg gcatccagaa atgcgtggcg gagcggcaga 3720
cgtgagccgg cacggcaggc ggcctcctcc tcctctcacg gcacggcagc tacgggggat 3780
tcctttccca ccgctccttc gctttccctt cctcgcccgc cgtaataaat agacaccccc 3840
tccacaccct ctttccccaa cctcgtgttg ttcggagcgc acacacacac aaccagatct 3900
cccccaaatc cacccgtcgg cacctccgct tcaaggtacg ccgctcgtcc tccccccccc 3960
cccctctcta ccttctctag atcggcgttc cggtccatgg ttagggcccg gtagttctac 4020
ttctgttcat gtttgtgtta gatccgtgtt tgtgttagat ccgtgctgct agcgttcgta 4080
cacggatgcg acctgtacgt cagacacgtt ctgattgcta acttgccagt gtttctcttt 4140
ggggaatcct gggatggctc tagccgttcc gcagacggga tcgatttcat gatttttttt 4200
gtttcgttgc atagggtttg gtttgccctt ttcctttatt tcaatatatg ccgtgcactt 4260
gtttgtcggg tcatcttttc atgctttttt ttgtcttggt tgtgatgatg tggtctggtt 4320
gggcggtcgt tctagatcgg agtagaattc tgtttcaaac tacctggtgg atttattaat 4380
tttggatctg tatgtgtgtg ccatacatat tcatagttac gaattgaaga tgatggatgg 4440
aaatatcgat ctaggatagg tatacatgtt gatgcgggtt ttactgatgc atatacagag 4500
atgctttttg ttcgcttggt tgtgatgatg tggtgtggtt gggcggtcgt tcattcgttc 4560
tagatcggag tagaatactg tttcaaacta cctggtgtat ttattaattt tggaactgta 4620
tgtgtgtgtc atacatcttc atagttacga gtttaagatg gatggaaata tcgatctagg 4680
ataggtatac atgttgatgt gggttttact gatgcatata catgatggca tatgcagcat 4740
ctattcatat gctctaacct tgagtaccta tctattataa taaacaagta tgttttataa 4800
ttattttgat cttgatatac ttggatgatg gcatatgcag cagctatatg tggatttttt 4860
tagccctgcc ttcatacgct atttatttgc ttggtactgt ttcttttgtc gatgctcacc 4920
ctgttgtttg gtgttacttc tgcaggccac catggcgcca aagaagaagc gcaaggtcga 4980
caagaagtac tccatcggcc tcgacatcgg caccaattct gttggctggg ccgtgatcac 5040
cgacgagtac aaggtgccgt ccaagaagtt caaggtcctc ggcaacaccg accgccactc 5100
catcaagaag aatctcatcg gcgccctgct gttcgactct ggcgagacag ccgaggctac 5160
aaggctcaag aggaccgcta gacgcaggta caccaggcgc aagaaccgca tctgctacct 5220
ccaagagatc ttctccaacg agatggccaa ggtggacgac agcttcttcc acaggctcga 5280
ggagagcttc ctcgtcgagg aggacaagaa gcacgagcgc catccgatct tcggcaacat 5340
cgtggatgag gtggcctacc acgagaagta cccgaccatc taccacctcc gcaagaagct 5400
cgtcgactcc accgataagg ccgacctcag gctcatctac ctcgccctcg cccacatgat 5460
caagttcagg ggccacttcc tcatcgaggg cgacctcaac ccggacaact ccgatgtgga 5520
caagctgttc atccagctcg tgcagaccta caaccagctg ttcgaggaga acccgatcaa 5580
cgcctctggc gttgacgcca aggctattct ctctgccagg ctctctaagt cccgcaagct 5640
cgagaatctg atcgcccaac ttccgggcga gaagaagaat ggcctcttcg gcaacctgat 5700
cgccctctct cttggcctca ccccgaactt caagtccaac ttcgacctcg ccgaggacgc 5760
caagctccag ctttccaagg acacctacga cgacgacctc gacaatctcc tcgcccagat 5820
tggcgatcag tacgccgatc tgttcctcgc cgccaagaat ctctccgacg ccatcctcct 5880
cagcgacatc ctcagggtga acaccgagat caccaaggcc ccactctccg cctccatgat 5940
caagaggtac gacgagcacc accaggacct cacactcctc aaggccctcg tgagacagca 6000
gctcccagag aagtacaagg agatcttctt cgaccagtcc aagaacggct acgccggcta 6060
catcgatggc ggcgcttctc aagaggagtt ctacaagttc atcaagccga tcctcgagaa 6120
gatggacggc accgaggagc tgctcgtgaa gctcaagaga gaggacctcc tccgcaagca 6180
gcgcaccttc gataatggct ccatcccgca ccagatccac ctcggcgagc ttcatgctat 6240
cctccgcagg caagaggact tctacccgtt cctcaaggac aaccgcgaga agattgagaa 6300
gatcctcacc ttccgcatcc cgtactacgt gggcccgctc gccaggggca actccaggtt 6360
cgcctggatg accagaaagt ccgaggagac aatcaccccc tggaacttcg aggaggtggt 6420
ggataagggc gcctctgccc agtctttcat cgagcgcatg accaacttcg acaagaacct 6480
cccgaacgag aaggtgctcc cgaagcactc actcctctac gagtacttca ccgtgtacaa 6540
cgagctgacc aaggtgaagt acgtgaccga ggggatgagg aagccagctt tccttagcgg 6600
cgagcaaaag aaggccatcg tcgacctgct gttcaagacc aaccgcaagg tgaccgtgaa 6660
gcagctcaag gaggactact tcaagaaaat cgagtgcttc gactccgtcg agatctccgg 6720
cgtcgaggat aggttcaatg cctccctcgg gacctaccac gacctcctca agattatcaa 6780
ggacaaggac ttcctcgaca acgaggagaa cgaggacatc ctcgaggaca tcgtgctcac 6840
cctcaccctc ttcgaggacc gcgagatgat cgaggagcgc ctcaagacat acgcccacct 6900
cttcgacgac aaggtgatga agcagctgaa gcgcaggcgc tataccggct ggggcaggct 6960
ctctaggaag ctcatcaacg gcatccgcga caagcagtcc ggcaagacga tcctcgactt 7020
cctcaagtcc gacggcttcg ccaaccgcaa cttcatgcag ctcatccacg acgactccct 7080
caccttcaag gaggacatcc aaaaggccca ggtgtccggc caaggcgatt ccctccatga 7140
acatatcgcc aatctcgccg gctccccggc tatcaagaag ggcattctcc agaccgtgaa 7200
ggtggtggac gagctggtga aggtgatggg caggcacaag ccagagaaca tcgtgatcga 7260
gatggcccgc gagaaccaga ccacacagaa gggccaaaag aactcccgcg agcgcatgaa 7320
gaggatcgag gagggcatta aggagctggg ctcccagatc ctcaaggagc acccagtcga 7380
gaacacccag ctccagaacg agaagctcta cctctactac ctccagaacg gccgcgacat 7440
gtacgtggac caagagctgg acatcaaccg cctctccgac tacgacgtgg accatattgt 7500
gccgcagtcc ttcctgaagg acgactccat cgacaacaag gtgctcaccc gctccgacaa 7560
gaacaggggc aagtccgata acgtgccgtc cgaagaggtc gtcaagaaga tgaagaacta 7620
ctggcgccag ctcctcaacg ccaagctcat cacccagagg aagttcgaca acctcaccaa 7680
ggccgagaga ggcggccttt ccgagcttga taaggccggc ttcatcaagc gccagctcgt 7740
cgagacacgc cagatcacaa agcacgtggc ccagatcctc gactcccgca tgaacaccaa 7800
gtacgacgag aacgacaagc tcatccgcga ggtgaaggtc atcaccctca agtccaagct 7860
cgtgtccgac ttccgcaagg acttccagtt ctacaaggtg cgcgagatca acaactacca 7920
ccacgcccac gacgcctacc tcaatgccgt ggtgggcaca gccctcatca agaagtaccc 7980
aaagctcgag tccgagttcg tgtacggcga ctacaaggtg tacgacgtgc gcaagatgat 8040
cgccaagtcc gagcaagaga tcggcaaggc gaccgccaag tacttcttct actccaacat 8100
catgaatttc ttcaagaccg agatcacgct cgccaacggc gagattagga agaggccgct 8160
catcgagaca aacggcgaga caggcgagat cgtgtgggac aagggcaggg atttcgccac 8220
agtgcgcaag gtgctctcca tgccgcaagt gaacatcgtg aagaagaccg aggttcagac 8280
cggcggcttc tccaaggagt ccatcctccc aaagcgcaac tccgacaagc tgatcgcccg 8340
caagaaggac tgggacccga agaagtatgg cggcttcgat tctccgaccg tggcctactc 8400
tgtgctcgtg gttgccaagg tcgagaaggg caagagcaag aagctcaagt ccgtcaagga 8460
gctgctgggc atcacgatca tggagcgcag cagcttcgag aagaacccaa tcgacttcct 8520
cgaggccaag ggctacaagg aggtgaagaa ggacctcatc atcaagctcc cgaagtacag 8580
cctcttcgag cttgagaacg gccgcaagag aatgctcgcc tctgctggcg agcttcagaa 8640
gggcaacgag cttgctctcc cgtccaagta cgtgaacttc ctctacctcg cctcccacta 8700
cgagaagctc aagggctccc cagaggacaa cgagcaaaag cagctgttcg tcgagcagca 8760
caagcactac ctcgacgaga tcatcgagca gatctccgag ttctccaagc gcgtgatcct 8820
cgccgatgcc aacctcgata aggtgctcag cgcctacaac aagcaccgcg ataagccaat 8880
tcgcgagcag gccgagaaca tcatccacct cttcaccctc accaacctcg gcgctccagc 8940
cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc tacacctcta ccaaggaggt 9000
tctcgacgcc accctcatcc accagtctat cacaggcctc tacgagacac gcatcgacct 9060
ctcacaactc ggcggcgatt caggcggctc cagcggcggc tctaagcgga ccgccgacgg 9120
atcagagttc gagagcccga agaagaagag gaaggtgtcc ggcggctcat ctggcggctc 9180
cacactcaat atcgaggacg agtacaggct gcatgagaca tccaaggagc ctgacgtctc 9240
cctcggcagc acatggctct cagatttccc acaggcctgg gccgagacag gcggcatggg 9300
cctcgccgtc cgccaggcgc cgctcatcat tccactgaag gcgacctcca caccggtgag 9360
catcaagcag tacccaatgt ctcaggaggc aaggctgggc atcaagccac acattcagag 9420
gctcctggac cagggcattc tggtgccttg ccagagcccg tggaacaccc ctctcctgcc 9480
ggtgaagaag cctggcacaa atgactaccg cccggtccag gatctcaggg aggtgaacaa 9540
gcgcgtcgag gatatccatc cgacagtccc gaacccatac aatctcctgt caggcctccc 9600
gccatctcac cagtggtaca ccgtgctcga cctgaaggat gcgttcttct gcctcaggct 9660
gcatccaaca agccagcctc tcttcgcctt cgagtggcgc gatccggaga tgggcatttc 9720
aggccagctc acctggacac ggctgccaca gggcttcaag aactctccta ccctcttcaa 9780
tgaggcgctc catcgggacc tggccgattt caggatccag cacccagacc tcattctcct 9840
ccagtatgtg gacgatctcc tgctcgccgc gacatccgag ctggattgcc agcagggaac 9900
ccgcgcgctg ctccagacac tgggaaatct gggatacagg gcatcagcga agaaggcaca 9960
gatctgccag aagcaggtca agtacctcgg ctacctgctc aaggagggac agaggtggct 10020
gacagaggca aggaaggaga cagtgatggg ccagcctacc ccgaagacac cacggcagct 10080
cagggagttc ctgggcaagg cgggcttctg ccgcctcttc atcccaggat tcgcggagat 10140
ggcggcgcca ctctaccctc tgaccaagcc tggcacactg ttcaactggg gaccagacca 10200
gcagaaggcg taccaggaga ttaagcaggc cctgctcaca gcacctgccc tcggcctgcc 10260
ggacctcaca aagccattcg agctgttcgt ggatgagaag cagggctacg cgaagggagt 10320
cctgacacag aagctgggac catggaggcg cccagtggcc tacctctcca agaagctgga 10380
cccagtggct gccggctggc ctccgtgcct gaggatggtg gcggccattg ccgtcctcac 10440
caaggatgcc ggcaagctga caatgggcca gcctctcgtc attctggcgc cgcatgcggt 10500
ggaggcgctc gtcaagcagc cacctgatag gtggctgtcc aacgcgcgca tgacccacta 10560
ccaggccctg ctcctggaca cagatagggt gcagttcggc ccagtggtcg ccctcaatcc 10620
tgccacactg ctgccactcc ctgaggaggg cctccagcat aactgcctcg atattctggc 10680
ggaggcccat ggaacccgcc ctgacctcac agatcagccg ctgccagacg ccgatcacac 10740
ctggtacaca gatggctcat ctctcctcca ggagggccag aggaaggccg gagccgcggt 10800
gaccacagag acagaggtca tctgggcaaa ggcgctccca gccggcacct ccgcacagag 10860
ggccgagctg attgcactga cacaggcgct caagatggcc gagggcaaga agctgaatgt 10920
gtacaccgac tcacgctacg ccttcgcgac agcccacatc catggagaga tctacaggag 10980
gaggggatgg ctcacatctg agggcaagga gatcaagaac aaggatgaga ttctcgcgct 11040
cctgaaggcc ctcttcctgc caaagcgcct gtcaatcatt cactgccctg gccatcagaa 11100
gggacactct gcggaggcaa ggggaaatag gatggccgac caggcggcca ggaaggcagc 11160
gatcaccgag acaccggata cctccacact cctgattgag aactccagcc catcaggcgg 11220
ctctaagagg accgccgacg gatcagagtt cgagagcccg aagaagaaga ggaaagtggg 11280
atcaggacca gccgccaaga gggtgaagct cgattgagag ctcgagctca agggtgggcg 11340
cgccgaccca gctttcttgt acaaagtggt gatatcccgc ggccatggcg gccgggagca 11400
tgcgacgtcg atctaactga ctagccgcgg ccatgctaga gtccgcaaaa atcaccagtc 11460
tctctctaca aatctatctc tctctatttt tctccagaat aatgtgtgag tagttcccag 11520
ataagggaat tagggttctt atagggtttc gctcatgtgt tgagcatata agaaaccctt 11580
agtatgtatt tgtatttgta aaatacttct atcaataaaa tttctaattc ctaaaaccaa 11640
aatccagtga cctggaattc gtaatcatgt catagctgtt tcctgtgtga aattgttatc 11700
cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 11760
aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 11820
acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 11880
ttggctagag cagcttgcca acatggtgga gcacgacact ctcgtctact ccaagaatat 11940
caaagataca gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc 12000
gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcaaaa ggacagtaga 12060
aaaggaaggt ggcacctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga 12120
tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca tcgtggaaaa 12180
agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgataaca tggtggagca 12240
cgacactctc gtctactcca agaatatcaa agatacagtc tcagaagacc aaagggctat 12300
tgagactttt caacaaaggg taatatcggg aaacctcctc ggattccatt gcccagctat 12360
ctgtcacttc atcaaaagga cagtagaaaa ggaaggtggc acctacaaat gccatcattg 12420
cgataaagga aaggctatcg ttcaagatgc ctctgccgac agtggtccca aagatggacc 12480
cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt 12540
ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca 12600
agaccttcct ctatataagg aagttcattt catttggaga ggacacgctg aaatcaccag 12660
tctctctcta caaatctatc tctctcgagc tttcgcagat cccggggggc aatgagatat 12720
gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 12780
cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 12840
aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 12900
ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 12960
ggagtttagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 13020
agacctgcct gaaaccgaac tgcccgctgt tctacaaccg gtcgcggagg ctatggatgc 13080
gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 13140
cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 13200
ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 13260
gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 13320
caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 13380
gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 13440
tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccacg 13500
actccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 13560
caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 13620
cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 13680
tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagaa 13740
atagagtaga tgccgaccgg atctgtcgat cgacaagctc gagtttctcc ataataatgt 13800
gtgagtagtt cccagataag ggaattaggg ttcctatagg gtttcgctca tgtgttgagc 13860
atataagaaa cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct 13920
aattcctaaa accaaaatcc agtactaaaa tccagatccc ccgaattaat tcggcgttaa 13980
ttcagtacat taaaaacgtc cgcaatgtgt tattaagttg tctaagcgtc aatttgttta 14040
caccacaata tatcctgcca ccagccagcc aacagctccc cgaccggcag ctcggcacaa 14100
aatcaccact cgatacaggc agcccatcag tccgggacgg cgtcagcggg agagccgttg 14160
taaggcggca gactttgctc atgttaccga tgctattcgg aagaacggca actaagctgc 14220
cgggtttgaa acacggatga tctcgcggag ggtagcatgt tgattgtaac gatgacagag 14280
cgttgctgcc tgtgatcacc gcggtttcaa aatcggctcc gtcgatacta tgttatacgc 14340
caactttgaa aacaactttg aaaaagctgt tttctggtat ttaaggtttt agaatgcaag 14400
gaacagtgaa ttggagttcg tcttgttata attagcttct tggggtatct ttaaatactg 14460
tagaaaagag gaaggaaata ataaatggct aaaatgagaa tatcaccgga attgaaaaaa 14520
ctgatcgaaa aataccgctg cgtaaaagat acggaaggaa tgtctcctgc taaggtatat 14580
aagctggtgg gagaaaatga aaacctatat ttaaaaatga cggacagccg gtataaaggg 14640
accacctatg atgtggaacg ggaaaaggac atgatgctat ggctggaagg aaagctgcct 14700
gttccaaagg tcctgcactt tgaacggcat gatggctgga gcaatctgct catgagtgag 14760
gccgatggcg tcctttgctc ggaagagtat gaagatgaac aaagccctga aaagattatc 14820
gagctgtatg cggagtgcat caggctcttt cactccatcg acatatcgga ttgtccctat 14880
acgaatagct tagacagccg cttagccgaa ttggattact tactgaataa cgatctggcc 14940
gatgtggatt gcgaaaactg ggaagaagac actccattta aagatccgcg cgagctgtat 15000
gattttttaa agacggaaaa gcccgaagag gaacttgtct tttcccacgg cgacctggga 15060
gacagcaaca tctttgtgaa agatggcaaa gtaagtggct ttattgatct tgggagaagc 15120
ggcagggcgg acaagtggta tgacattgcc ttctgcgtcc ggtcgatcag ggaggatatc 15180
ggggaagaac agtatgtcga gctatttttt gacttactgg ggatcaagcc tgattgggag 15240
aaaataaaat attatatttt actggatgaa ttgttttagt acctagaatg catgaccaaa 15300
atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 15360
tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 15420
ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 15480
ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 15540
cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 15600
gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 15660
gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 15720
acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 15780
gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 15840
agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 15900
tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 15960
agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 16020
cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 16080
gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 16140
ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact 16200
ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac 16260
gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 16320
cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 16380
gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gggtgccttg atgtgggcgc 16440
cggcggtcga gtggcgacgg cgcggcttgt ccgcgccctg gtagattgcc tggccgtagg 16500
ccagccattt ttgagcggcc agcggccgcg ataggccgac gcgaagcggc ggggcgtagg 16560
gagcgcagcg accgaagggt aggcgctttt tgcagctctt cggctgtgcg ctggccagac 16620
agttatgcac aggccaggcg ggttttaaga gttttaataa gttttaaaga gttttaggcg 16680
gaaaaatcgc cttttttctc ttttatatca gtcacttaca tgtgtgaccg gttcccaatg 16740
tacggctttg ggttcccaat gtacgggttc cggttcccaa tgtacggctt tgggttccca 16800
atgtacgtgc tatccacagg aaacagacct tttcgacctt tttcccctgc tagggcaatt 16860
tgccctagca tctgctccgt acattaggaa ccggcggatg cttcgccctc gatcaggttg 16920
cggtagcgca tgactaggat cgggccagcc tgccccgcct cctccttcaa atcgtactcc 16980
ggcaggtcat ttgacccgat cagcttgcgc acggtgaaac agaacttctt gaactctccg 17040
gcgctgccac tgcgttcgta gatcgtcttg aacaaccatc tggcttctgc cttgcctgcg 17100
gcgcggcgtg ccaggcggta gagaaaacgg ccgatgccgg gatcgatcaa aaagtaatcg 17160
gggtgaaccg tcagcacgtc cgggttcttg ccttctgtga tctcgcggta catccaatca 17220
gctagctcga tctcgatgta ctccggccgc ccggtttcgc tctttacgat cttgtagcgg 17280
ctaatcaagg cttcaccctc ggataccgtc accaggcggc cgttcttggc cttcttcgta 17340
cgctgcatgg caacgtgcgt ggtgtttaac cgaatgcagg tttctaccag gtcgtctttc 17400
tgctttccgc catcggctcg ccggcagaac ttgagtacgt ccgcaacgtg tggacggaac 17460
acgcggccgg gcttgtctcc cttcccttcc cggtatcggt tcatggattc ggttagatgg 17520
gaaaccgcca tcagtaccag gtcgtaatcc cacacactgg ccatgccggc cggccctgcg 17580
gaaacctcta cgtgcccgtc tggaagctcg tagcggatca cctcgccagc tcgtcggtca 17640
cgcttcgaca gacggaaaac ggccacgtcc atgatgctgc gactatcgcg ggtgcccacg 17700
tcatagagca tcggaacgaa aaaatctggt tgctcgtcgc ccttgggcgg cttcctaatc 17760
gacggcgcac cggctgccgg cggttgccgg gattctttgc ggattcgatc agcggccgct 17820
tgccacgatt caccggggcg tgcttctgcc tcgatgcgtt gccgctgggc ggcctgcgcg 17880
gccttcaact tctccaccag gtcatcaccc agcgccgcgc cgatttgtac cgggccggat 17940
ggtttgcgac cgctcacgcc gattcctcgg gcttgggggt tccagtgcca ttgcagggcc 18000
ggcagacaac ccagccgctt acgcctggcc aaccgcccgt tcctccacac atggggcatt 18060
ccacggcgtc ggtgcctggt tgttcttgat tttccatgcc gcctccttta gccgctaaaa 18120
ttcatctact catttattca tttgctcatt tactctggta gctgcgcgat gtattcagat 18180
agcagctcgg taatggtctt gccttggcgt accgcgtaca tcttcagctt ggtgtgatcc 18240
tccgccggca actgaaagtt gacccgcttc atggctggcg tgtctgccag gctggccaac 18300
gttgcagcct tgctgctgcg tgcgctcgga cggccggcac ttagcgtgtt tgtgcttttg 18360
ctcattttct ctttacctca ttaactcaaa tgagttttga tttaatttca gcggccagcg 18420
cctggacctc gcgggcagcg tcgccctcgg gttctgattc aagaacggtt gtgccggcgg 18480
cggcagtgcc tgggtagctc acgcgctgcg tgatacggga ctcaagaatg ggcagctcgt 18540
acccggccag cgcctcggca acctcaccgc cgatgcgcgt gcctttgatc gcccgcgaca 18600
cgacaaaggc cgcttgtagc cttccatccg tgacctcaat gcgctgctta accagctcca 18660
ccaggtcggc ggtggcccat atgtcgtaag ggcttggctg caccggaatc agcacgaagt 18720
cggctgcctt gatcgcggac acagccaagt ccgccgcctg gggcgctccg tcgatcacta 18780
cgaagtcgcg ccggccgatg gccttcacgt cgcggtcaat cgtcgggcgg tcgatgccga 18840
caacggttag cggttgatct tcccgcacgg ccgcccaatc gcgggcactg ccctggggat 18900
cggaatcgac taacagaaca tcggccccgg cgagttgcag ggcgcgggct agatgggttg 18960
cgatggtcgt cttgcctgac ccgcctttct ggttaagtac agcgataacc ttcatgcgtt 19020
ccccttgcgt atttgtttat ttactcatcg catcatatac gcagcgaccg catgacgcaa 19080
gctgttttac tcaaatacac atcacctttt tagacggcgg cgctcggttt cttcagcggc 19140
caagctggcc ggccaggccg ccagcttggc atcagacaaa ccggccagga tttcatgcag 19200
ccgcacggtt gagacgtgcg cgggcggctc gaacacgtac ccggccgcga tcatctccgc 19260
ctcgatctct tcggtaatga aaaacggttc gtcctggccg tcctggtgcg gtttcatgct 19320
tgttcctctt ggcgttcatt ctcggcggcc gccagggcgt cggcctcggt caatgcgtcc 19380
tcacggaagg caccgcgccg cctggcctcg gtgggcgtca cttcctcgct gcgctcaagt 19440
gcgcggtaca gggtcgagcg atgcacgcca agcagtgcag ccgcctcttt cacggtgcgg 19500
ccttcctggt cgatcagctc gcgggcgtgc gcgatctgtg ccggggtgag ggtagggcgg 19560
gggccaaact tcacgcctcg ggccttggcg gcctcgcgcc cgctccgggt gcggtcgatg 19620
attagggaac gctcgaactc ggcaatgccg gcgaacacgg tcaacaccat gcggccggcc 19680
ggcgtggtgg tgtcggccca cggctctgcc aggctacgca ggcccgcgcc ggcctcctgg 19740
atgcgctcgg caatgtccag taggtcgcgg gtgctgcggg ccaggcggtc tagcctggtc 19800
actgtcacaa cgtcgccagg gcgtaggtgg tcaagcatcc tggccagctc cgggcggtcg 19860
cgcctggtgc cggtgatctt ctcggaaaac agcttggtgc agccggccgc gtgcagttcg 19920
gcccgttggt tggtcaagtc ctggtcgtcg gtgctgacgc gggcatagcc cagcaggcca 19980
gcggcggcgc tcttgttcat ggcgtaatgt ctccggttct agtcgcaagt attctacttt 20040
atgcgactaa aacacgcgac aagaaaacgc caggaaaagg gcagggcggc agcctgtcgc 20100
gtaacttagg acttgtgcga catgtcgttt tcagaagacg gctgcactga acgtcagaag 20160
ccgactgcac tatagcagcg gaggggttgg atcaaagtac tttgatcccg aggggaaccc 20220
tgtggttggc atgcacatac aaatggacga acggataaac cttttcacgc ccttttaaat 20280
atccgttatt ctaa 20294
<210> 2
<211> 19240
<212> DNA
<213> pegRNA
<400> 2
taaacgctct tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta 60
aactgaaggc gggaaacgac aatctgatcc aagctcaagc tgctctagca ttcgccattc 120
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 180
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 240
cgacgttgta aaacgacggc cagtgccaag cttaagggat ctttaaacat acgaacagat 300
cacttaaagt tcttctgaag caacttaaag ttatcaggca tgcatggatc ttggaggaat 360
cagatgtgca gtcagggacc atagcacaag acaggcgtct tctactggtg ctaccagcaa 420
atgctggaag ccgggaacac tgggtacgtt ggaaaccacg tgatgtgaag aagtaagata 480
aactgtagga gaaaagcatt tcgtagtggg ccatgaagcc tttcaggaca tgtattgcag 540
tatgggccgg cccattacgc aattggacga caacaaagac tagtattagt accacctcgg 600
ctatccacat agatcaaagc tgatttaaaa gagttgtgca gatgatccgt ggcatgagac 660
caacccagtg gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct 720
cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt 780
tcatggcttg ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc 840
aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc 900
agggcagtcg ccctaaaaca aagttaaaca tcatggggga agcggtgatc gccgaagtat 960
cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg 1020
ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt 1080
tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc 1140
ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca 1200
ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg 1260
gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg 1320
atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg 1380
cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct 1440
taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt 1500
tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg 1560
actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg 1620
cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg 1680
tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta gctagaaatt 1740
cgttcaagcc gacgccgctt cgcggcgcgg cttaactcaa gcgttagatg cactaagcac 1800
ataattgctc acagccaaac tatcaggtca agtctgcttt tattattttt aagcgtgcat 1860
aataagccgg tctcattttt tttagtaaag cttgatatcg aattcctgca gtgcagcgtg 1920
acccggtcgt gcccctctct agagataatg agcattgcat gtctaagtta taaaaaatta 1980
ccacatattt tttttgtcac acttgtttga agtgcagttt atctatcttt atacatatat 2040
ttaaacttta ctctacgaat aatataatct atagtactac aataatatca gtgttttaga 2100
gaatcatata aatgaacagt tagacatggt ctaaaggaca attgagtatt ttgacaacag 2160
gactctacag ttttatcttt ttagtgtgca tgtgttctcc tttttttttg caaatagctt 2220
cacctatata atacttcatc cattttatta gtacatccat ttagggttta gggttaatgg 2280
tttttataga ctaatttttt tagtacatct attttattct attttagcct ctaaattaag 2340
aaaactaaaa ctctatttta gtttttttat ttaataattt agatataaaa tagaataaaa 2400
taaagtgact aaaaattaaa caaataccct ttaagaaatt aaaaaaacta aggaaacatt 2460
tttcttgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt ctaacggaca 2520
ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca cggcatctct 2580
gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg ctccgctgtc 2640
ggcatccaga aatgcgtggc ggagcggcag acgtgagccg gcacggcagg cggcctcctc 2700
ctcctctcac ggcacggcag ctacggggga ttcctttccc accgctcctt cgctttccct 2760
tcctcgcccg ccgtaataaa tagacacccc ctccacaccc tctttcccca acctcgtgtt 2820
gttcggagcg cacacacaca caaccagatc tcccccaaat ccacccgtcg gcacctccgc 2880
ttcaaggtac gccgctcgtc ctcccccccc ccccctctct accttctcta gatcggcgtt 2940
ccggtccatg gttagggccc ggtagttcta cttctgttca tgtttgtgtt agatccgtgt 3000
ttgtgttaga tccgtgctgc tagcgttcgt acacggatgc gacctgtacg tcagacacgt 3060
tctgattgct aacttgccag tgtttctctt tggggaatcc tgggatggct ctagccgttc 3120
cgcagacggg atcgatttca tgattttttt tgtttcgttg catagggttt ggtttgccct 3180
tttcctttat ttcaatatat gccgtgcact tgtttgtcgg gtcatctttt catgcttttt 3240
tttgtcttgg ttgtgatgat gtggtctggt tgggcggtcg ttctagatcg gagtagaatt 3300
ctgtttcaaa ctacctggtg gatttattaa ttttggatct gtatgtgtgt gccatacata 3360
ttcatagtta cgaattgaag atgatggatg gaaatatcga tctaggatag gtatacatgt 3420
tgatgcgggt tttactgatg catatacaga gatgcttttt gttcgcttgg ttgtgatgat 3480
gtggtgtggt tgggcggtcg ttcattcgtt ctagatcgga gtagaatact gtttcaaact 3540
acctggtgta tttattaatt ttggaactgt atgtgtgtgt catacatctt catagttacg 3600
agtttaagat ggatggaaat atcgatctag gataggtata catgttgatg tgggttttac 3660
tgatgcatat acatgatggc atatgcagca tctattcata tgctctaacc ttgagtacct 3720
atctattata ataaacaagt atgttttata attattttga tcttgatata cttggatgat 3780
ggcatatgca gcagctatat gtggattttt ttagccctgc cttcatacgc tatttatttg 3840
cttggtactg tttcttttgt cgatgctcac cctgttgttt ggtgttactt ctgcagcccg 3900
ggggatcccc aatacttgta tggccgcggc cgcgccacca tggccccaaa gaagaagcgc 3960
aaggtcgaca agaagtactc catcggcctc gacatcggca ccaattctgt tggctgggcc 4020
gtgatcaccg acgagtacaa ggtgccgtcc aagaagttca aggtcctcgg caacaccgac 4080
cgccactcca tcaagaagaa tctcatcggc gccctgctgt tcgactctgg cgagacagcc 4140
gaggctacaa ggctcaagag gaccgctaga cgcaggtaca ccaggcgcaa gaaccgcatc 4200
tgctacctcc aagagatctt ctccaacgag atggccaagg tggacgacag cttcttccac 4260
aggctcgagg agagcttcct cgtcgaggag gacaagaagc acgagcgcca tccgatcttc 4320
ggcaacatcg tggatgaggt ggcctaccac gagaagtacc cgaccatcta ccacctccgc 4380
aagaagctcg tcgactccac cgataaggcc gacctcaggc tcatctacct cgccctcgcc 4440
cacatgatca agttcagggg ccacttcctc atcgagggcg acctcaaccc ggacaactcc 4500
gatgtggaca agctgttcat ccagctcgtg cagacctaca accagctgtt cgaggagaac 4560
ccgatcaacg cctctggcgt tgacgccaag gctattctct ctgccaggct ctctaagtcc 4620
cgcaggctcg agaatctgat cgcccaactt ccgggcgaga agaagaatgg cctcttcggc 4680
aacctgatcg ccctctctct tggcctcacc ccgaacttca agtccaactt cgacctcgcc 4740
gaggacgcca agctccagct ttccaaggac acctacgacg acgacctcga caatctcctc 4800
gcccagattg gcgatcagta cgccgatctg ttcctcgccg ccaagaatct ctccgacgcc 4860
atcctcctca gcgacatcct cagggtgaac accgagatca ccaaggcccc actctccgcc 4920
tccatgatca agaggtacga cgagcaccac caggacctca cactcctcaa ggccctcgtg 4980
agacagcagc tcccagagaa gtacaaggag atcttcttcg accagtccaa gaacggctac 5040
gccggctaca tcgatggcgg cgcttctcaa gaggagttct acaagttcat caagccgatc 5100
ctcgagaaga tggacggcac cgaggagctg ctcgtgaagc tcaatagaga ggacctcctc 5160
cgcaagcagc gcaccttcga taatggctcc atcccgcacc agatccacct cggcgagctt 5220
catgctatcc tccgcaggca agaggacttc tacccgttcc tcaaggacaa ccgcgagaag 5280
attgagaaga tcctcacctt ccgcatcccg tactacgtgg gcccgctcgc caggggcaac 5340
tccaggttcg cctggatgac cagaaagtcc gaggagacaa tcaccccctg gaacttcgag 5400
gaggtggtgg ataagggcgc ctctgcccag tctttcatcg agcgcatgac caacttcgac 5460
aagaacctcc cgaacgagaa ggtgctcccg aagcactcac tcctctacga gtacttcacc 5520
gtgtacaacg agctgaccaa ggtgaagtac gtgaccgagg ggatgaggaa gccagctttc 5580
cttagcggcg agcaaaagaa ggccatcgtc gacctgctgt tcaagaccaa ccgcaaggtg 5640
accgtgaagc agctcaagga ggactacttc aagaaaatcg agtgcttcga ctccgtcgag 5700
atctccggcg tcgaggatag gttcaatgcc tccctcggga cctaccacga cctcctcaag 5760
attatcaagg acaaggactt cctcgacaac gaggagaacg aggacatcct cgaggacatc 5820
gtgctcaccc tcaccctctt cgaggaccgc gagatgatcg aggagcgcct caagacatac 5880
gcccacctct tcgacgacaa ggtgatgaag cagctgaagc gcaggcgcta taccggctgg 5940
ggcaggctct ctaggaagct catcaacggc atccgcgaca agcagtccgg caagacgatc 6000
ctcgacttcc tcaagtccga cggcttcgcc aaccgcaact tcatgcagct catccacgac 6060
gactccctca ccttcaagga ggacatccaa aaggcccagg tgtccggcca aggcgattcc 6120
ctccatgaac atatcgccaa tctcgccggc tccccggcta tcaagaaggg cattctccag 6180
accgtgaagg tggtggacga gctggtgaag gtgatgggca ggcacaagcc agagaacatc 6240
gtgatcgaga tggcccgcga gaaccagacc acacagaagg gccaaaagaa ctcccgcgag 6300
cgcatgaaga ggatcgagga gggcattaag gagctgggct cccagatcct caaggagcac 6360
ccagtcgaga acacccagct ccagaacgag aagctctacc tctactacct ccagaacggc 6420
cgcgacatgt acgtggacca agagctggac atcaaccgcc tctccgacta cgacgtggac 6480
catattgtgc cgcagtcctt cctgaaggac gactccatcg acaacaaggt gctcacccgc 6540
tccgacaaga acaggggcaa gtccgataac gtgccgtccg aagaggtcgt caagaagatg 6600
aagaactact ggcgccagct cctcaacgcc aagctcatca cccagaggaa gttcgacaac 6660
ctcaccaagg ccgagagagg cggcctttcc gagcttgata aggccggctt catcaagcgc 6720
cagctcgtcg agacacgcca gatcacaaag cacgtggccc agatcctcga ctcccgcatg 6780
aacaccaagt acgacgagaa cgacaagctc atccgcgagg tgaaggtcat caccctcaag 6840
tccaagctcg tgtccgactt ccgcaaggac ttccagttct acaaggtgcg cgagatcaac 6900
aactaccacc acgcccacga cgcctacctc aatgccgtgg tgggcacagc cctcatcaag 6960
aagtacccaa agctcgagtc cgagttcgtg tacggcgact acaaggtgta cgacgtgcgc 7020
aagatgatcg ccaagtccga gcaagagatc ggcaaggcga ccgccaagta cttcttctac 7080
tccaacatca tgaatttctt caagaccgag atcacgctcg ccaacggcga gattaggaag 7140
aggccgctca tcgagacaaa cggcgagaca ggcgagatcg tgtgggacaa gggcagggat 7200
ttcgccacag tgcgcaaggt gctctccatg ccgcaagtga acatcgtgaa gaagaccgag 7260
gttcagaccg gcggcttctc caaggagtcc atcctcccaa agcgcaactc cgacaagctg 7320
atcgcccgca agaaggactg ggacccgaag aagtatggcg gcttcgattc tccgaccgtg 7380
gcctactctg tgctcgtggt tgccaaggtc gagaagggca agagcaagaa gctcaagtcc 7440
gtcaaggagc tgctgggcat cacgatcatg gagcgcagca gcttcgagaa gaacccaatc 7500
gacttcctcg aggccaaggg ctacaaggag gtgaagaagg acctcatcat caagctcccg 7560
aagtacagcc tcttcgagct tgagaacggc cgcaagagaa tgctcgcctc tgctggcgag 7620
cttcagaagg gcaacgagct tgctctcccg tccaagtacg tgaacttcct ctacctcgcc 7680
tcccactacg agaagctcaa gggctcccca gaggacaacg agcaaaagca gctgttcgtc 7740
gagcagcaca agcactacct cgacgagatc atcgagcaga tctccgagtt ctccaagcgc 7800
gtgatcctcg ccgatgccaa cctcgataag gtgctcagcg cctacaacaa gcaccgcgat 7860
aagccaattc gcgagcaggc cgagaacatc atccacctct tcaccctcac caacctcggc 7920
gctccagccg ccttcaagta cttcgacacc accatcgacc gcaagcgcta cacctctacc 7980
aaggaggttc tcgacgccac cctcatccac cagtctatca caggcctcta cgagacacgc 8040
atcgacctct cacaactcgg cggcgattcc ggcggctcca gcggcggctc atctggatca 8100
gagacaccag gcacatcaga gtcagcaaca ccggagtcca gcggcggctc atctggcggc 8160
tccagcacac tcaatatcga ggacgagtac aggctgcatg agacatccaa ggagcctgac 8220
gtctccctcg gcagcacatg gctctcagat ttcccacagg cctgggccga gacaggcggc 8280
atgggcctcg ccgtccgcca ggcgccgctc atcattccac tgaaggcgac ctccacaccg 8340
gtgagcatca agcagtaccc aatgtctcag gaggcaaggc tgggcatcaa gccacacatt 8400
cagaggctcc tggaccaggg cattctggtg ccttgccaga gcccgtggaa cacccctctc 8460
ctgccggtga agaagcctgg cacaaatgac taccgcccgg tccaggatct cagggaggtg 8520
aacaagcgcg tcgaggatat ccatccgaca gtcccgaacc catacaatct cctgtcaggc 8580
ctcccgccat ctcaccagtg gtacaccgtg ctcgacctga aggatgcgtt cttctgcctc 8640
aggctgcatc caacaagcca gcctctcttc gccttcgagt ggcgcgatcc ggagatgggc 8700
atttcaggcc agctcacctg gacacggctg ccacagggct tcaagaactc tcctaccctc 8760
ttcaatgagg cgctccatcg ggacctggcc gatttcagga tccagcaccc agacctcatt 8820
ctcctccagt atgtggacga tctcctgctc gccgcgacat ccgagctgga ttgccagcag 8880
ggaacccgcg cgctgctcca gacactggga aatctgggat acagggcatc agcgaagaag 8940
gcacagatct gccagaagca ggtcaagtac ctcggctacc tgctcaagga gggacagagg 9000
tggctgacag aggcaaggaa ggagacagtg atgggccagc ctaccccgaa gacaccacgg 9060
cagctcaggg agttcctggg caaggcgggc ttctgccgcc tcttcatccc aggattcgcg 9120
gagatggcgg cgccactcta ccctctgacc aagcctggca cactgttcaa ctggggacca 9180
gaccagcaga aggcgtacca ggagattaag caggccctgc tcacagcacc tgccctcggc 9240
ctgccggacc tcacaaagcc attcgagctg ttcgtggatg agaagcaggg ctacgcgaag 9300
ggagtcctga cacagaagct gggaccatgg aggcgcccag tggcctacct ctccaagaag 9360
ctggacccag tggctgccgg ctggcctccg tgcctgagga tggtggcggc cattgccgtc 9420
ctcaccaagg atgccggcaa gctgacaatg ggccagcctc tcgtcattct ggcgccgcat 9480
gcggtggagg cgctcgtcaa gcagccacct gataggtggc tgtccaacgc gcgcatgacc 9540
cactaccagg ccctgctcct ggacacagat agggtgcagt tcggcccagt ggtcgccctc 9600
aatcctgcca cactgctgcc actccctgag gagggcctcc agcataactg cctcgatatt 9660
ctggcggagg cccatggaac ccgccctgac ctcacagatc agccgctgcc agacgccgat 9720
cacacctggt acacagatgg ctcatctctc ctccaggagg gccagaggaa ggccggagcc 9780
gcggtgacca cagagacaga ggtcatctgg gcaaaggcgc tcccagccgg cacctccgca 9840
cagagggccg agctgattgc actgacacag gcgctcaaga tggccgaggg caagaagctg 9900
aatgtgtaca ccgactcacg ctacgccttc gcgacagccc acatccatgg agagatctac 9960
aggaggaggg gatggctcac atctgagggc aaggagatca agaacaagga tgagattctc 10020
gcgctcctga aggccctctt cctgccaaag cgcctgtcaa tcattcactg ccctggccat 10080
cagaagggac actctgcgga ggcaagggga aataggatgg ccgaccaggc ggccaggaag 10140
gcagcgatca ccgagacacc ggatacctcc acactcctga ttgagaactc cagcccatca 10200
ggcggctcta agaggaccgc cgacggatca gagttcgagc cgaagaagaa gaggaaggtg 10260
tccggcggct ccccgaagaa gaagaggaag gtgtccggcg gctccccgaa gaagaagagg 10320
aaagtgtgag agctccggcc gggagcatgc gacgtcgatc taactgacta gccgcggcca 10380
tgctagagtc cgcaaaaatc accagtctct ctctacaaat ctatctctct ctatttttct 10440
ccagaataat gtgtgagtag ttcccagata agggaattag ggttcttata gggtttcgct 10500
catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc 10560
aataaaattt ctaattccta aaaccaaaat ccagtgacct gaattcgtaa tcatgtcata 10620
gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 10680
cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 10740
ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 10800
acgcgcgggg agaggcggtt tgcgtattgg ctagagcagc ttgccaacat ggtggagcac 10860
gacactctcg tctactccaa gaatatcaaa gatacagtct cagaagacca aagggctatt 10920
gagacttttc aacaaagggt aatatcggga aacctcctcg gattccattg cccagctatc 10980
tgtcacttca tcaaaaggac agtagaaaag gaaggtggca cctacaaatg ccatcattgc 11040
gataaaggaa aggctatcgt tcaagatgcc tctgccgaca gtggtcccaa agatggaccc 11100
ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg 11160
gattgatgtg ataacatggt ggagcacgac actctcgtct actccaagaa tatcaaagat 11220
acagtctcag aagaccaaag ggctattgag acttttcaac aaagggtaat atcgggaaac 11280
ctcctcggat tccattgccc agctatctgt cacttcatca aaaggacagt agaaaaggaa 11340
ggtggcacct acaaatgcca tcattgcgat aaaggaaagg ctatcgttca agatgcctct 11400
gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac 11460
gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga cgtaagggat 11520
gacgcacaat cccactatcc ttcgcaagac cttcctctat ataaggaagt tcatttcatt 11580
tggagaggac acgctgaaat caccagtctc tctctacaaa tctatctctc tcgagctttc 11640
gcagatcccg gggggcaatg agatatgaaa aagcctgaac tcaccgcgac gtctgtcgag 11700
aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa 11760
gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc 11820
tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc 11880
ccgattccgg aagtgcttga cattggggag tttagcgaga gcctgaccta ttgcatctcc 11940
cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttcta 12000
caaccggtcg cggaggctat ggatgcgatc gctgcggccg atcttagcca gacgagcggg 12060
ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc 12120
gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg 12180
tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg 12240
cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca 12300
gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc 12360
ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg 12420
catccggagc ttgcaggatc gccacgactc cgggcgtata tgctccgcat tggtcttgac 12480
caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga 12540
tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga 12600
agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc 12660
cccagcactc gtccgagggc aaagaaatag agtagatgcc gaccggatct gtcgatcgac 12720
aagctcgagt ttctccataa taatgtgtga gtagttccca gataagggaa ttagggttcc 12780
tatagggttt cgctcatgtg ttgagcatat aagaaaccct tagtatgtat ttgtatttgt 12840
aaaatacttc tatcaataaa atttctaatt cctaaaacca aaatccagta ctaaaatcca 12900
gatcccccga attaattcgg cgttaattca gtacattaaa aacgtccgca atgtgttatt 12960
aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca 13020
gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc catcagtccg 13080
ggacggcgtc agcgggagag ccgttgtaag gcggcagact ttgctcatgt taccgatgct 13140
attcggaaga acggcaacta agctgccggg tttgaaacac ggatgatctc gcggagggta 13200
gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg atcaccgcgg tttcaaaatc 13260
ggctccgtcg atactatgtt atacgccaac tttgaaaaca actttgaaaa agctgttttc 13320
tggtatttaa ggttttagaa tgcaaggaac agtgaattgg agttcgtctt gttataatta 13380
gcttcttggg gtatctttaa atactgtaga aaagaggaag gaaataataa atggctaaaa 13440
tgagaatatc accggaattg aaaaaactga tcgaaaaata ccgctgcgta aaagatacgg 13500
aaggaatgtc tcctgctaag gtatataagc tggtgggaga aaatgaaaac ctatatttaa 13560
aaatgacgga cagccggtat aaagggacca cctatgatgt ggaacgggaa aaggacatga 13620
tgctatggct ggaaggaaag ctgcctgttc caaaggtcct gcactttgaa cggcatgatg 13680
gctggagcaa tctgctcatg agtgaggccg atggcgtcct ttgctcggaa gagtatgaag 13740
atgaacaaag ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg ctctttcact 13800
ccatcgacat atcggattgt ccctatacga atagcttaga cagccgctta gccgaattgg 13860
attacttact gaataacgat ctggccgatg tggattgcga aaactgggaa gaagacactc 13920
catttaaaga tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc gaagaggaac 13980
ttgtcttttc ccacggcgac ctgggagaca gcaacatctt tgtgaaagat ggcaaagtaa 14040
gtggctttat tgatcttggg agaagcggca gggcggacaa gtggtatgac attgccttct 14100
gcgtccggtc gatcagggag gatatcgggg aagaacagta tgtcgagcta ttttttgact 14160
tactggggat caagcctgat tgggagaaaa taaaatatta tattttactg gatgaattgt 14220
tttagtacct agaatgcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 14280
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 14340
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 14400
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 14460
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 14520
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 14580
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 14640
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 14700
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 14760
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 14820
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 14880
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 14940
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 15000
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 15060
cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg 15120
gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 15180
gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc 15240
aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 15300
tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 15360
gaggcagggt gccttgatgt gggcgccggc ggtcgagtgg cgacggcgcg gcttgtccgc 15420
gccctggtag attgcctggc cgtaggccag ccatttttga gcggccagcg gccgcgatag 15480
gccgacgcga agcggcgggg cgtagggagc gcagcgaccg aagggtaggc gctttttgca 15540
gctcttcggc tgtgcgctgg ccagacagtt atgcacaggc caggcgggtt ttaagagttt 15600
taataagttt taaagagttt taggcggaaa aatcgccttt tttctctttt atatcagtca 15660
cttacatgtg tgaccggttc ccaatgtacg gctttgggtt cccaatgtac gggttccggt 15720
tcccaatgta cggctttggg ttcccaatgt acgtgctatc cacaggaaac agaccttttc 15780
gacctttttc ccctgctagg gcaatttgcc ctagcatctg ctccgtacat taggaaccgg 15840
cggatgcttc gccctcgatc aggttgcggt agcgcatgac taggatcggg ccagcctgcc 15900
ccgcctcctc cttcaaatcg tactccggca ggtcatttga cccgatcagc ttgcgcacgg 15960
tgaaacagaa cttcttgaac tctccggcgc tgccactgcg ttcgtagatc gtcttgaaca 16020
accatctggc ttctgccttg cctgcggcgc ggcgtgccag gcggtagaga aaacggccga 16080
tgccgggatc gatcaaaaag taatcggggt gaaccgtcag cacgtccggg ttcttgcctt 16140
ctgtgatctc gcggtacatc caatcagcta gctcgatctc gatgtactcc ggccgcccgg 16200
tttcgctctt tacgatcttg tagcggctaa tcaaggcttc accctcggat accgtcacca 16260
ggcggccgtt cttggccttc ttcgtacgct gcatggcaac gtgcgtggtg tttaaccgaa 16320
tgcaggtttc taccaggtcg tctttctgct ttccgccatc ggctcgccgg cagaacttga 16380
gtacgtccgc aacgtgtgga cggaacacgc ggccgggctt gtctcccttc ccttcccggt 16440
atcggttcat ggattcggtt agatgggaaa ccgccatcag taccaggtcg taatcccaca 16500
cactggccat gccggccggc cctgcggaaa cctctacgtg cccgtctgga agctcgtagc 16560
ggatcacctc gccagctcgt cggtcacgct tcgacagacg gaaaacggcc acgtccatga 16620
tgctgcgact atcgcgggtg cccacgtcat agagcatcgg aacgaaaaaa tctggttgct 16680
cgtcgccctt gggcggcttc ctaatcgacg gcgcaccggc tgccggcggt tgccgggatt 16740
ctttgcggat tcgatcagcg gccgcttgcc acgattcacc ggggcgtgct tctgcctcga 16800
tgcgttgccg ctgggcggcc tgcgcggcct tcaacttctc caccaggtca tcacccagcg 16860
ccgcgccgat ttgtaccggg ccggatggtt tgcgaccgct cacgccgatt cctcgggctt 16920
gggggttcca gtgccattgc agggccggca gacaacccag ccgcttacgc ctggccaacc 16980
gcccgttcct ccacacatgg ggcattccac ggcgtcggtg cctggttgtt cttgattttc 17040
catgccgcct cctttagccg ctaaaattca tctactcatt tattcatttg ctcatttact 17100
ctggtagctg cgcgatgtat tcagatagca gctcggtaat ggtcttgcct tggcgtaccg 17160
cgtacatctt cagcttggtg tgatcctccg ccggcaactg aaagttgacc cgcttcatgg 17220
ctggcgtgtc tgccaggctg gccaacgttg cagccttgct gctgcgtgcg ctcggacggc 17280
cggcacttag cgtgtttgtg cttttgctca ttttctcttt acctcattaa ctcaaatgag 17340
ttttgattta atttcagcgg ccagcgcctg gacctcgcgg gcagcgtcgc cctcgggttc 17400
tgattcaaga acggttgtgc cggcggcggc agtgcctggg tagctcacgc gctgcgtgat 17460
acgggactca agaatgggca gctcgtaccc ggccagcgcc tcggcaacct caccgccgat 17520
gcgcgtgcct ttgatcgccc gcgacacgac aaaggccgct tgtagccttc catccgtgac 17580
ctcaatgcgc tgcttaacca gctccaccag gtcggcggtg gcccatatgt cgtaagggct 17640
tggctgcacc ggaatcagca cgaagtcggc tgccttgatc gcggacacag ccaagtccgc 17700
cgcctggggc gctccgtcga tcactacgaa gtcgcgccgg ccgatggcct tcacgtcgcg 17760
gtcaatcgtc gggcggtcga tgccgacaac ggttagcggt tgatcttccc gcacggccgc 17820
ccaatcgcgg gcactgccct ggggatcgga atcgactaac agaacatcgg ccccggcgag 17880
ttgcagggcg cgggctagat gggttgcgat ggtcgtcttg cctgacccgc ctttctggtt 17940
aagtacagcg ataaccttca tgcgttcccc ttgcgtattt gtttatttac tcatcgcatc 18000
atatacgcag cgaccgcatg acgcaagctg ttttactcaa atacacatca cctttttaga 18060
cggcggcgct cggtttcttc agcggccaag ctggccggcc aggccgccag cttggcatca 18120
gacaaaccgg ccaggatttc atgcagccgc acggttgaga cgtgcgcggg cggctcgaac 18180
acgtacccgg ccgcgatcat ctccgcctcg atctcttcgg taatgaaaaa cggttcgtcc 18240
tggccgtcct ggtgcggttt catgcttgtt cctcttggcg ttcattctcg gcggccgcca 18300
gggcgtcggc ctcggtcaat gcgtcctcac ggaaggcacc gcgccgcctg gcctcggtgg 18360
gcgtcacttc ctcgctgcgc tcaagtgcgc ggtacagggt cgagcgatgc acgccaagca 18420
gtgcagccgc ctctttcacg gtgcggcctt cctggtcgat cagctcgcgg gcgtgcgcga 18480
tctgtgccgg ggtgagggta gggcgggggc caaacttcac gcctcgggcc ttggcggcct 18540
cgcgcccgct ccgggtgcgg tcgatgatta gggaacgctc gaactcggca atgccggcga 18600
acacggtcaa caccatgcgg ccggccggcg tggtggtgtc ggcccacggc tctgccaggc 18660
tacgcaggcc cgcgccggcc tcctggatgc gctcggcaat gtccagtagg tcgcgggtgc 18720
tgcgggccag gcggtctagc ctggtcactg tcacaacgtc gccagggcgt aggtggtcaa 18780
gcatcctggc cagctccggg cggtcgcgcc tggtgccggt gatcttctcg gaaaacagct 18840
tggtgcagcc ggccgcgtgc agttcggccc gttggttggt caagtcctgg tcgtcggtgc 18900
tgacgcgggc atagcccagc aggccagcgg cggcgctctt gttcatggcg taatgtctcc 18960
ggttctagtc gcaagtattc tactttatgc gactaaaaca cgcgacaaga aaacgccagg 19020
aaaagggcag ggcggcagcc tgtcgcgtaa cttaggactt gtgcgacatg tcgttttcag 19080
aagacggctg cactgaacgt cagaagccga ctgcactata gcagcggagg ggttggatca 19140
aagtactttg atcccgaggg gaaccctgtg gttggcatgc acatacaaat ggacgaacgg 19200
ataaaccttt tcacgccctt ttaaatatcc gttattctaa 19240

Claims (9)

1.一种用于骨干载体的pegRNA表达框，其特征在于，所述pegRNA表达框包含启动子、tRNA基因序列、壮观霉素抗性基因SpR、EQ序列、RNA核酶HDV序列和终止子，其中，所述tRNA基因的核苷酸序列如Seq ID No.1第1274至1345位所示，所述壮观霉素抗性基因SpR的核苷酸序列如Seq ID No.1第1452至2558位所示，所述EQ序列的核苷酸序列如Seq ID No.1第2566至2607位所示，RNA核酶HDV的核苷酸序列如Seq ID No.1第2608至2675位所示，所述pegRNA表达框的核苷酸序列如序列表Seq ID No.1的第274至2954位所示，所述壮观霉素抗性基因SpR用于替换为靶向目的DNA片段的sgRNA、sgRNA骨架序列、逆转录模板RT和引物结合位点PBS、8 bp linker序列。
2.根据权利要求1所述的用于骨干载体的pegRNA表达框，其特征在于，其中，所述启动子为 35S-CmYLCV-U6复合启动子，所述终止子为polyT-HSPt复合终止子。
3.一种用于植物引导编辑系统的骨干载体，其特征在于，其包含权利要求1所述的pegRNA表达框和融合蛋白表达框。
4. 根据权利要求3所述的骨干载体，其特征在于，所述融合蛋白表达框包括ZmUBI启动子、经改造后的Cas9切刻酶编码序列、逆转录酶M-MLV RT编码序列和35s终止子，其中，ZmUBI启动子的核苷酸序列如Seq ID No.1第2961至4939位所示，Cas9切刻酶的核苷酸序列如Seq ID No.1第4979至9079位所示，逆转录酶M-MLV RT的核苷酸序列如Seq ID No.1第9182至11254位所示，35s终止子的核苷酸序列如Seq ID No.1第11389至11653位所示。
5. 根据权利要求4所述的骨干载体，其特征在于，融合蛋白表达框中，在Cas9切刻酶的5’端存在一个核定位信号 SV40 NLS，其核苷酸序列如Seq ID No.1第4958至4978位所示；Cas9切刻酶与逆转录酶M-MLV RT编码序列之间含有33aa的连接序列，其核苷酸序列如SeqID No.1第9080至9181位所示；M-MLV RT编码序列的3’端含有核定位信号 SV40 NLS和CYNLS，其核苷酸序列如Seq ID No.1第11258至11314位所示。
6. 根据权利要求5所述的骨干载体，其特征在于，所述骨干质粒载体还包括T-DNA的左、右边界序列，其中所述左边界的核苷酸序列如Seq ID No.1第14035至14060位所示，所述右边界的核苷酸序列如Seq ID No.1第1至26位所示；所述向导pegRNA表达框和所述融合蛋白表达框位于所述左边界和所述右边界之间。
7.利用权利要求3-6中任意一项所述的骨干载体构建重组载体的方法，其特征在于，所述方法包括：
按照目的基因的编码序列和突变类型，选择sgRNA序列，得到相应的逆转录模板RT和引物结合位点PBS序列，用BsaI内切酶切开权利要求3-6之一所述的骨干载体，利用含BsaI的Golden Gate反应，将sgRNA序列、sgRNA骨架序列、RT和PBS序列、8 bp linker序列替换壮观霉素抗性基因，形成用于作物目的基因的引导编辑重组载体。
8.根据权利要求7所述的方法，其特征在于，所述方法包括将所述重组载体转入植物细胞，使细胞同时含有针对靶标基因的pegRNA和融合蛋白；并对生物体的基因组进行编辑，获得生物突变体，所述基因组序列的编辑为基因组序列的碱基替换、缺失和插入。
9.一种植物引导编辑系统，其特征在于，所述植物引导编辑系统包含权利要求1或2所述的pegRNA表达框或者权利要求3-6之一所述的骨干载体。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111378051A (zh) * 2020-03-25 2020-07-07 北京市农林科学院 Pe-p2引导编辑系统及其在基因组碱基编辑中的应用
CN113201557A (zh) * 2021-05-10 2021-08-03 安徽省农业科学院水稻研究所 一种引导编辑系统介导作物产生内源除草剂抗性的方法
WO2021165508A1 (en) * 2020-02-21 2021-08-26 Biogemma Prime editing technology for plant genome engineering
CN113564164A (zh) * 2021-07-19 2021-10-29 中国农业大学 一种提高先导编辑效率的载体和方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106811479B (zh) * 2015-11-30 2019-10-25 中国农业科学院作物科学研究所 利用CRISPR/Cas9系统定点修饰ALS基因获得抗除草剂水稻的系统及其应用
AU2021236683A1 (en) * 2020-03-19 2022-11-17 Intellia Therapeutics, Inc. Methods and compositions for directed genome editing

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021165508A1 (en) * 2020-02-21 2021-08-26 Biogemma Prime editing technology for plant genome engineering
CN111378051A (zh) * 2020-03-25 2020-07-07 北京市农林科学院 Pe-p2引导编辑系统及其在基因组碱基编辑中的应用
CN113201557A (zh) * 2021-05-10 2021-08-03 安徽省农业科学院水稻研究所 一种引导编辑系统介导作物产生内源除草剂抗性的方法
CN113564164A (zh) * 2021-07-19 2021-10-29 中国农业大学 一种提高先导编辑效率的载体和方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Engineered pegRNAs improve prime editing efficiency;Nelson等;Nat Biotechnol.;第40卷(第3期);第402–410页 *
Enhanced prime editing systems by manipulating cellular determinants of editing outcomes;Chen PJ等;Cell;第184卷(第22期);第5635-5652页 *

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