一种可调节的先导编辑器及其应用

1.本发明属于生物医药领域，涉及一种可调节的先导编辑器及其应用，具体涉及的可调节的先 导编辑器(prime editors):snpes和snipes技术属于基因治疗的平台技术，可以用于定点突 变，插入，筛除基因组dna片段等疾病的基因治疗。


背景技术：

2.人类的疾病大多与基因密切相关，所以根据基因进行针对性的改变一直是生命科学的长期愿 望。尽管基因编辑技术取得了快速进展，但与疾病相关的75000多个人类已知的遗传变异中，大 多数疾病还是存在难以纠正的问题。
3.利用工程性核酸酶例如zfps,talens或者crispr-cas可以有效的利用dna双链断裂(doublestrand breaks,dsbs)介导的dna编辑方式，在特定基因组位点进行插入与缺失。但是，dsb的 产生会导致许多不良的结果，比如编辑不单一，基因序列易位和p53的激活。
4.在细胞体内，染色质经常发生dna双链断裂(dna double-strand breaks,dsbs)一方面是 在减数分裂重组和脊椎动物免疫系统发育过程中所必需的，另一方面，dsb的错误修复会产生突 变并促进基因组不稳定，无法修复dsbs会导致细胞死亡。通常来说，有两种主要的修复机制会 被dsb所激活，一种是同源性介导修复(homology-directed repair，hdr),另一种是非同源末 端连接(non-homologous end joining，nhej)。作为dna双链断裂最主要的修复通路，hdr使 用同源序列作为修复的模板以完成基因序列的校正，并且只能发生在细胞g2/s期；相反，nhej 在修复过程不需要同源序列就能在dsb附近的基因组位点引入插入和缺失。但是，由于同源重组 机制本身的局限性，hdr介导的基因校正效率一直都很低(通常小于5％)。因此，极大的限制了 crispr/cas基因编辑工具从科研向应用的转变。应运而生的碱基编辑器(base editor,be)， 胞嘧啶和腺苷碱基编辑器可将c
·
g转化为t
·
a，将a
·
t转化为g
·
c，该方法虽然能极大的提 高基因位点的编辑效率，但是该方法突变的方式单一并且需要dna双链断裂，对于精准治疗方面 的应用，这些方法都具有一定的局限性。
5.先导编辑(prime editing)是在2019年底由mit david liu实验室发明的。这项技术被认 为是基于crispr的第三代基因编辑技术，可以对基因组中的任何位点进行精确地点突变、小片 段dna的插入和缺失。目前先导编辑的主要问题是编辑效率在不同的细胞，不同的位点差异很 大。所以优化先导编辑的效率迫在眉睫。


技术实现要素：

6.先导编辑(prime editing)可以在没有双链断裂的情况下定点突变，插入，筛除基因组dna 片段。先导编辑器(prime editors)中pegrna的稳定性和错误折叠会影响先导编辑的效率。与 受cas9蛋白保护的sgrna相比，pegrna的3
‘
延伸部分可能暴露在细胞中，更容易受到核酸外 切酶的降解。我们已经申请一个相关专利，通过在pegrna的3'末端加入rna
颈环结构并通过让 pegrna的3'末端结合到cas9,开发了tpes来提高编辑效率。本发明在tpes的此基础上，把 pegrna一分为二变成sgrna和先导rna(prime rna)，开发了snpes,方便对rna的化学合成与 修饰。同时也开发了可诱导的snipes用于实现对先导编辑在时间和空间上的控制。本发明通过 将3
‘
延伸部分从pegrna分离开，并在分离开的3’延伸部分加上不同rna适配子或者将3
‘
延 伸部分进行环化，以保护3
‘
延伸部分不易降解且保证了cas9与sgrna结合的稳定性。并且我 们开发了可诱导型的先导编辑工具。
7.本发明的目的之一是提供一种基因先导编辑系统，包含载体，所述载体含有编码sgrna的 基因、编码分离的先导rna的基因以及编码融合蛋白的基因，所述分离的先导rna包含逆转 录模板rtt、主要结合位点pbs、rna茎环结构。
8.所述融合蛋白为rna结合蛋白rbp、cas9切口酶或其变体、逆转录酶的融合蛋白；或，所 述融合蛋白包括：包含rna结合蛋白rbp与第一诱导结合分子的第一融合蛋白，以及包含cas9 切口酶或其变体、逆转录酶、第二诱导结合分子的第二融合蛋白片段。
9.所述rna茎环结构选自以下至少一项：
10.1)序列分别如seq id no.1～5所示的ms2、pp7、combs、csy4bs、boxb；
11.2)在1)的5
′
端和/或3
′
端添加一个或几个核苷酸得到的rna序列；
12.3)与1)或2)的rna序列具有85％以上的同一性的rna序列。
13.所述rna茎环结构连接于所述主要结合位点pbs的3’端，和/或逆转录模板rtt的5
’ꢀ
端；
14.所述分离的先导rna从5’端至3’端依次为逆转录模板rtt、主要结合位点pbs和rna 茎环结构；
15.所述分离的先导rna从5’端至3’端依次为rna茎环结构、逆转录模板rtt、主要结合 位点pbs；
16.所述分离的先导rna为线性或环形；
17.所述rbp为特异性结合rna茎环结构的蛋白；所述rbp为一个或多个；
18.所述载体为一个或多个；
19.所述编码sgrna的基因、编码分离的先导rna的基因以及编码融合蛋白的基因为一个或 多个；
20.所述编码sgrna的基因、编码分离的先导rna的基因以及编码融合蛋白的基因位于同一 载体或不同载体上；
21.所述第一诱导结合分子、第二诱导结合分子为适于通过诱导分子进行结合的分子；
22.所述第一诱导结合分子位于rbp的n端或c端；
23.所述第二融合蛋白片段由n端至c端依次包括第二诱导结合分子、cas9或其变体、逆转录 酶；或，所述第二融合蛋白片段由n端至c端依次包括cas9或其变体、逆转录酶、第二诱导结 合分子。
24.所述rbp选自tdmcp、tdpcp、com、csy4h29a、n22p中的任一种；
25.所述cas9切口酶为h840a；
26.所述逆转录酶为mmlv；
27.所述第一诱导分子、第二诱导分子分别独立地选自两对蛋白fkbp和frb或者abi和
pyl1 中的一种；即，当第一诱导分子为fkbp(seq id no.13)时，第二诱导分子为frb(seq idno.14)；当第一诱导分子为frb(seq id no.14)时，第二诱导分子为fkbp(seq id no.13)； 当第一诱导分子为abi(seq id no.12)时，第二诱导分子为pyl1(seq id no.11)；当第一 诱导分子为pyl1(seq id no.11)时，第二诱导分子为abi(seq id no.12)。
28.所述系统还含有筛选标记蛋白。
29.本发明的另一目的是提供一种分离的先导rna，所述分离的先导为如上任一项所述的系统 中所述的分离的先导rna。
30.本发明的另一目的是提供一种融合蛋白，所述融合蛋白包含rna结合蛋白rbp与第一诱导 结合分子，或者，所述融合蛋白包含cas9切口酶或其变体、逆转录酶和第二诱导结合分子。
31.所述第一诱导结合分子、第二诱导结合分子为适于通过诱导分子进行结合的分子；
32.所述第一诱导分子、第二诱导分子分别独立地选自两对蛋白fkbp和frb或者abi和pyl1 中的一种；即，当第一诱导分子为fkbp(seq id no.13)时，第二诱导分子为frb(seq idno.14)；当第一诱导分子为frb(seq id no.14)时，第二诱导分子为fkbp(seq id no.13)； 当第一诱导分子为abi(seq id no.12)时，第二诱导分子为pyl1(seq id no.11)；当第一 诱导分子为pyl1(seq id no.11)时，第二诱导分子为abi(seq id no.12)。
33.所述cas9切口酶为h840a；
34.所述逆转录酶为mmlv。
35.本发明的另一目的是提供一种核酸，所述核酸编码如上所述的分离的先导rna和/或融合蛋 白。
36.本发明的另一目的是提供一种构建体，所述构建体含有如上所述的核酸。
37.本发明的另一目的是提供一种一种宿主细胞，所述宿主细胞包含如上所述的构建体或基因组 中整合有外源的如上所述的核酸。
38.如上任一项所述的系统、分离的先导rna、融合蛋白、核酸、构建体或宿主细胞在以下至 少一项中的应用：
39.1)用于生物体或生物细胞基因组序列的编辑；优选地，用于碱基替换；
40.2)用于制备生物体或生物细胞基因组序列的编辑的产品；
41.3)用于提高生物体或生物细胞基因组序列的编辑效率；
42.4)用于制备提高生物体或生物细胞基因组序列的编辑效率的产品。
43.用于dna编辑或rna编辑；优选地，用于dna编辑；进一步优选地，用于runx1_+5 位点的编辑；更进一步优选地，用于runx1_+5g
·
c到t
·
a的编辑。
44.本发明另一目的是提供一种基因编辑的方法，包括将如上任一项所述的系统、分离的先导 rna、融合蛋白、核酸、构建体或宿主细胞导入待编辑体中实现基因的编辑。
附图说明
45.图1.使用不同适配体连接到cas9的snpe编辑效率的比较。
46.图2.拆分-pegrna通过将引导rna与cas9-h840a结合来维持pe活性。
47.图3.(a)带有com、boxb或csy4适配体的snpe示意图。snpe-com、-boxb或-csy4由
t2a-gfp、 pcmv-csy4h29a-pe2-t2a-gfp和pcmv-tdpcp-pe2-t2a-gfp质粒。
79.诱导型编辑的表达质粒是在pcmv-pe2-t2a-gfp质粒基础之上，将合成的frb，abi蛋白克隆 进去，分别得到pcmv-frb-pe2-t2a-gfp和pcmv-abi-pe2-t2a-gfp(补充序列s1)。pcmv-fkbp
‑ꢀ
mcp和pcmv-pyl1-mcp(补充序列s1)在pcmv-mcp质粒的基础上，将fkbp和pyl1蛋白克隆 得到。
80.步骤1.2.1中质粒构建实验中扩增各种片段使用pcr体系如下：
81.表1.2.1.1 pcr扩增体系
[0082][0083]
表1.2.1.2 pcr程序
[0084][0085]
表1.2.1.3退火程序
[0086][0087]
表1.2.1.4同源重组反应
[0088]
[0089][0090]
表1.2.1.5 sgrna golden gate组装反应
[0091][0092]
1.2.2细胞培养与转染和基因组抽提
[0093]
hek293ft(thermo fisher scientific)、hela(atcc,ccl-2)和u2os(atcc,htb-96)细胞在含有 高葡萄糖的10％fbs(胎牛血清，thermo fisher scientific)的dmem培养基中培养。所有细胞均 在37℃、5％co2的加湿培养箱中培养。转染前，将细胞接种在12孔板中，细胞密度大约在60％ 左右，并使用lipofectamine 2000(thermo fisher scientific)进行转染。为了测量基因编辑效率，在 1.5ml离心管中，加入50μl opti-dmem，1.5μg prime editor 2(pe2,dcas9-mmlv)或rbp-pe2， 以及500ng pe-all-rna、spe-all-rna或tpe-all-rna表达质粒。涡旋使其充分混匀，另取一个1.5 ml离心管，加入50μl opti-dmem和4μl lipofectamine 2000转染试剂，静置5min。将2管溶 液合并，混匀后静置15min共转染到hek293ft、u2os或hela细胞。转染后72小时，利用 流式细胞术(bd facs aria)收集prime编辑器(gfp+)和pe-all-rna、spe-all-rna、tpe-all-rna双 阳性细胞。
[0094]
1.2.3基因组dna抽提和pcr扩增
[0095]
使用quickextract dna extraction solution(lucigen)按照试剂操作说明提取细胞的基因组，将 抽提得到的基因组dna利用以下引物(补充表2)进行pcr扩增。
[0096]
1.2.4深度测序
[0097]
使用包含truseq接头的基因座特异性引物从基因组dna中扩增感兴趣的基因组位点。合 并等量的pcr产物并利用胶回收试剂盒凝胶进行纯化。使用双端150bp illuminaminiseq运行对纯化的文库进行深度测序。
[0098]
图7.ppb-snpe-all-rna包含三种类型的prna：psnpe-5'-prna、psnpe-3'-prna和psnpe-circ
‑ꢀ
prna。psnpe-5'-prna由5'-末端的茎环(深绿色)、rtt(金色)和pbs(洋红色)组成。psnpe
‑ꢀ
3'-prna由3'-末端包含茎环(深绿色)、rtt(金色)和pbs(洋红色)组成。psnpe-circ-prna 包含位于两端(紫色)、茎环(深绿色)、rtt(金色)和pbs(洋红色)的环状序列。psnpe-sgrna 包含sgrna(蓝色)，ppe3-sgrna包含sgrnanick(橙色)，pdonor5.1由ccdb基因(绿色)、嘌 呤霉素(黄色)、p2a(紫色)和tagbfp2(青色)组成。
[0099]
ms2(seq id no.1)
[0100]
acatgaggatcacccatgt
[0101]
pp7(seq id no.2)
[0102]
ggagcagacgatatggcgtcgctcc
[0103]
com(com蛋白结合位点)(seq id no.3)
[0104]
ctgaatgcctgcgagcatc
[0105]
csy4(csy4蛋白结合位点)(seq id no.4)
[0106]
actgccgtataggcagctaagaaa
[0107]
boxb(boxb rna结合位点)(seq id no.5)
[0108]
ggccctgaagaagggcc
[0109]
mcp(ms2外壳蛋白)(seq id no.6)
[0110]
masnftqfvlvdnggtgdvtvapsnfangiaewissnsrsqaykvtcsvrqssaqnrkytikvevpkgawrsylnm eltipifatnsdcelivkamqgllkdgnpipsaiaansgiy
[0111]
pcp(pp7外壳蛋白)(seq id no.7)
[0112]
mgsktivlsvgeatrtlteiqstadrqifeekvgplvgrlrltaslrqngaktayrvnlkldqadvvdsglpkvrytqv wshdvtivansteasrkslydltkslvatsqvedlvvnlvplgr
[0113]
com(com rna结合蛋白)(seq id no.8)
[0114]
gsmksirckncnkllfkadsfdhieircprckrhiimlnacehptekhcgkrekithsdetvrygstsghklnggggg mdaksltaws
[0115]
csy4h29a(csy4结合蛋白h29a突变体)(seq id no.9)
[0116]
mdhyldirlrpdpefppaqlmsvlfgklaqalvaqggdrigvsfpdldesrsrlgerlrihasaddlrallarpwleg lrdhlqfgepavvphptpyrqvsrvqaksnperlrrrlmrrhdlseeearkripdtvaraldlpfvtlrsqstgqhfr lfirhgplqvtaeeggftcyglskggfvpwf
[0117]
n22p(λn22肽段结合蛋白)(seq id no.10)
[0118]
gnartrrrerraekqaqwkaan
[0119]
pyl1-mcp(吡拉菌素结合脱落酸受体-ms2外壳蛋白融合蛋白)(seq id no.11)
[0120]
gggaptqdeftqlsqsiaefhtyqlgngrcssllaqrihappetvwsvvrrfdrpqiykhfi kscnvsedfemrvgctrdvnvisglpantsrerldlldddrrvtgfsitggehrlrnyksvt tvhrfekeeeeeriwtvvlesyvvdvpegnseedtrlfadtvirlnlqklasiteamnkrtad gsefespkkkrkvmasnftqfvlvdnggtgdvtvapsnfangiaewissnsrsqaykvtcsv rqssaqnrkytikvevpkgawrsylnmeltipifatnsdcelivkamqgllkdgnpipsaiaan sgiy
[0121]
abi(2c型蛋白磷酸酶)(seq id no.12)
[0122]
trvplygftsicgrrpemeaavstiprflqsssgsmldgrfdpqsaahffgvydghggsqv anycre
rmhlalaeeiakekpmlcdgdtwlekwkkalfnsflrvdseiesvapetvgstsvvavvfpshifvancgdsravlcrgktalplsvdhkpdredeaarieaaggkviqwngarvfgvlamsrsigdrylkpsiipdpevtavkrvkeddclilasdgvwdvmtdeeacemarkrillwhkknavagdaslladerrkegkdpaamsaaeylsklaiqrgskdnisvvvvdlk
[0123]
fkbp-mcp(rap介导fk506结合蛋白12-ms2外壳蛋白融合蛋白)(seqidno.13)
[0124]
mgvqvetispgdgrtfpkrgqtcvvhytgmledgkkfdssrdrnkpfkfmlgkqevirgweegvaqmsvgqrakltispdyaygatghpgiipphatlvfdvellklekrtadgsefespkkkrkvmasnftqfvlvdnggtgdvtvapsnfangiaewissnsrsqaykvtcsvrqssaqnrkytikvevpkgawrsylnmeltipifatnsdcelivkamqgllkdgnpipsaiaansgiy
[0125]
frb(fkbp12-rapamycin复合物的结合位点)(seqidno.14)
[0126]
ilwhemwhegleeasrlyfgernvkgmfevleplhammergpqtlketsfnqaygrdlmeaqewcrkymksgnvkdltqawdlyyhvfrrisk
[0127]
补充表格1
[0128]
ppe-pegrna
[0129]
jak2-f:
[0130]
ataattgaagacataccgacctgtgactcatgaaacacgtttgagagctagaaatagcaagttcaaataaggc(seqidno.15)
[0131]
jak2-r:ataattgaagacgcaaaactgtgactcatgaaatacaggaagaatgtgcaccgactcggtgcc(seqidno.16)
[0132]
hdac1-f:
[0133]
ataattgaagacataccggcaccatgcaaagaagtccggtttgagagctagaaatagcaagttcaaataaggc(seqidno.17)
[0134]
hdac1-r:ataattgaagacgcaaaatgcaaagaagtgcgaggcatctggctgcaccgactcggtgcc(seqidno.18)
[0135]
eed-f:
[0136]
ataattgaagacataccgccggttgttgcaatcttacggtttgagagctagaaatagcaagttcaaataaggc(seqidno.19)
[0137]
eed-r:ataattgaagacgcaaaagttgttgcaatctttcgtggatgctgatgcaccgactcggtgcc(seqidno.20)
[0138]
bcl11a-f:
[0139]
ataattgaagacataccgtcacagataaacttctgcacgtttgagagctagaaatagcaagttcaaataaggc(seqidno.21)
[0140]
bcl11a-r:
[0141]
ataattgaagacgcaaaacacagataaacttctgaactggaggggccgcaccgactcggtgcc(seqidno.22)
[0142]
srd5a3-f:
[0143]
ataattgaagacataccgccttactcaatctctgttccgtttgagagctagaaatagcaagttcaaataaggc(seqidno.23)
[0144]
srd5a3-r:
[0145]
ataattgaagacgcaaaattactcaatctctgttactgggagcaccttttcgcaccgactcggtgcc(seqidno.24)
[0146]
srd5a1-f:
[0147]
ataattgaagacataccgtccattcagatcatatcctagtttgagagctagaaatagcaagttcaaataaggc(seqidno.25)
[0148]
srd5a1-r:
[0149]
ataattgaagacgcaaaacattcagatcatatcataaggaatctcagaaagcaccgactcggtgcc(seqidno.26)
[0150]
dyrk1a-f:ataattgaagacataccgagcacatcaaggacattctagtttgagagctagaaatagcaagttcaaataaggc(seqidno.27)
[0151]
dyrk1a-r:ataattgaagacgcaaaacaaggacattgtaaggatgatgcaccgactcggtgcc(seqidno.28)
[0152]
dmd-f:
[0153]
ataattgaagacataccgctacctctgtgatactcttcgtttgagagctagaaatagcaagttcaaataaggc(seqidno.29)
[0154]
dmd-r:ataattgaagacgcaaaaacctctgtgatactcctcaggtgcacctgcaccgactcggtgcc(seqidno.30)
[0155]
runx1-f:ataattgaagacataccggcattttcaggaggaagcgagtttgagagctagaaatagcaagttcaaataaggc(seqidno.31)
[0156]
runx1-r:ataattgaagacgcaaaaattttcaggaggaagcgattgcttcagacagcaccgactcggtgcc(seqidno.32)
[0157]
gfap-f:ataattgaagacataccgagcctgtgtccatataaagggttttagagctagaaatagcaagttaaaataaggctagtc(seqidno.33)
[0158]
gfap-r:ataattgaagacgcaaaatgtgtccatataatggaggagttggaagcaccgactcggtgcc(seqidno.34)
[0159]
pspe-pegrna
[0160]
jak2-ms2-f:
[0161]
ataattgaagacataccgacctgtgactcatgaaacacgtttgagagctagaaatagcaagttcaaataaggc(seqidno.35)
[0162]
jak2-ms2-r:
[0163]
ataattgaagacgcaaaaacatgggtgatcctcatgtctgtgactcatgaaatacaggaagaatgtgcaccgactcggtgcc(seqidno.36)
[0164]
hdac1-ms2-f:
[0165]
ataattgaagacataccggcaccatgcaaagaagtccggtttgagagctagaaatagcaagttcaaataaggc(seqidno.37)
[0166]
hdac1-ms2-r:
[0167]
ataattgaagacgcaaaaacatgggtgatcctcatgttgcaaagaagtgcgaggcatctggctgcaccgactcggtgcc(seqidno.38)
[0168]
eed-ms2-f:
[0169]
ataattgaagacataccgccggttgttgcaatcttacggtttgagagctagaaatagcaagttcaaataaggc(seqidno.39)
[0170]
eed-ms2-r:
[0171]
ataattgaagacgcaaaaacatgggtgatcctcatgtgttgttgcaatctttcgtggatgctgatgcaccgactcggtgcc(seqidno.40)
[0172]
bcl11a-ms2-f:
[0173]
ataattgaagacataccgtcacagataaacttctgcacgtttgagagctagaaatagcaagttcaaataaggc(seqidno.41)
[0174]
bcl11a-ms2-r:
[0175]
ataattgaagacgcaaaaacatgggtgatcctcatgtcacagataaacttctgaactggaggggccgcaccgactcggtgcc(seqidno.42)
[0176]
srd5a3-ms2-f:
[0177]
ataattgaagacataccgccttactcaatctctgttccgtttgagagctagaaatagcaagttcaaataaggc(seqidno.43)
[0178]
srd5a3-ms2-r:
[0179]
ataattgaagacgcaaaaacatgggtgatcctcatgtttactcaatctctgttactgggagcaccttttcgcaccgactcggtgcc(seqidno.44)
[0180]
srd5a1-ms2-f:
[0181]
ataattgaagacataccgtccattcagatcatatcctagtttgagagctagaaatagcaagttcaaataaggc(seqidno.45)
[0182]
srd5a1-ms2-r:
[0183]
ataattgaagacgcaaaaacatgggtgatcctcatgtcattcagatcatatcataaggaatctcagaaagcaccgactcggtgcc(seqidno.46)
[0184]
dyrk1a-ms2-f:
[0185]
ataattgaagacataccgagcacatcaaggacattctagtttgagagctagaaatagcaagttcaaataaggc(seqidno.47)
[0186]
dyrk1a-ms2-r:
[0187]
ataattgaagacgcaaaaacatgggtgatcctcatgtcaaggacattgtaaggatgatgcaccgactcggtgcc(seqidno.48)
[0188]
dmd-ms2-f:
[0189]
ataattgaagacataccgctacctctgtgatactcttcgtttgagagctagaaatagcaagttcaaataaggc(seqidno.49)
[0190]
dmd-ms2-r:
[0191]
ataattgaagacgcaaaaacatgggtgatcctcatgtacctctgtgatactcctcaggtgcacctgcaccgactcggtgcc(seqidno.50)
[0192]
runx1-ms2-f：
[0193]
ataattgaagacataccggcattttcaggaggaagcgagtttgagagctagaaatagcaagttcaaataaggc(seqidno.51)
[0194]
runx1-ms2-r:ataattgaagacgcaaaaacatgggtgatcctcatgtattttcaggaggaagcgat
tgcttcagacagcaccgactcggtgcc(seqidno.52)
[0195]
gfap-ms2-f:
[0196]
ataattgaagacataccgagcctgtgtccatataaagggttttagagctagaaatagcaagttaaaataaggctagtc(seqidno.53)
[0197]
gfap-ms2-r:
[0198]
ataattgaagacgcaaaaacatgggtgatcctcatgttgtgtccatataatggaggagttggaagcaccgactcggtgcc(seqidno.54)
[0199]
runx1-com-f:
[0200]
ataattgaagacataccggcattttcaggaggaagcgagtttgagagctagaaatagcaagttcaaataaggc(seqidno.55)
[0201]
runx1-com-r:
[0202]
ataattgaagacgcaaaagatgctcgcaggcattcagattttcaggaggaagcgattgcttcagacagcaccgactcggtgcc(seqidno.56)
[0203]
runx1-pp7-f:
[0204]
ataattgaagacataccggcattttcaggaggaagcgagtttgagagctagaaatagcaagttcaaataaggc(seqidno.57)
[0205]
runx1-pp7-r:
[0206]
ataattgaagacgcaaaaggagcgacgccatatcgtctgctccattttcaggaggaagcgattgcttcagacagcaccgactcggtgcc(seqidno.58)
[0207]
runx1-csy4-f:
[0208]
ataattgaagacataccggcattttcaggaggaagcgagtttgagagctagaaatagcaagttcaaataaggc(seqidno.59)
[0209]
runx1-csy4-r：
[0210]
ataattgaagacgcaaaactgcctatacggcagtgaacattttcaggaggaagcgattgcttcagacagcaccgactcggtgcc(seqidno.60)
[0211]
ppe3-sgrna
[0212]
jak2-f:accgtggggtttgatcgttttctt(seqidno.61)jak2-r:aaacaagaaaacgatcaaacccca(seqidno.62)
[0213]
hdac1-f:accgatactttagcagttccagg(seqidno.63)hdac1-r:aaactcctggaactgctaaagtat(seqidno.64)
[0214]
eed-f:accgtgattaatttataaaagttt(seqidno.65)eed-r:aaacaaacttttataaattaatca(seqidno.66)
[0215]
bcl11a-f:accgctgcactcatcccaggcgtg(seqidno.67)bcl11a-r:aaaccacgcctgggatgagtgcag(seqidno.68)
[0216]
srd5a3-f:accgccagtaacagagattgagta(seqidno.69)srd5a3-r:aaactactcaatctctgttactgg(seqidno.70)
[0217]
srd5a1-f:accgtccttatgatatgatctgaa(seqidno.71)srd5a1-r:aaacttcagatcatatcataagga(seqidno.72)
[0218]
dyrk1a-f:accgaagagtgaggagaaggcagg(seqidno.73)dyrk1a-r:
aaaccctgccttctcctcactctt(seq id no.74)
[0219]
dmd-f:accggtcaatgctctccttttcac(seq id no.75)dmd
‑ꢀ
r:aaacgtgaaaaggagagcattgac(seq id no.76)
[0220]
runx1-f:accgcatgtactcacctctcatga(seq id no.77)runx1-r: aaactcatgagaggtgagtacatg(seq id no.78)
[0221]
补充表格2
[0222]
gene
[0223]
jak2-f:ctacacgacgctcttccgatctgtggcggcatgattttgt(seq id no.79)jak2-r: agacgtgtgctcttccgatctccaagcattttagataag(seq id no.80)
[0224]
hdac1-f:ctacacgacgctcttccgatcttcatgcccagcaagtgctgtgaaac(seq id no.81)hdac1
‑ꢀ
r:agacgtgtgctcttccgatctgacactcaccgagcacatcctagcc(seq id no.82)
[0225]
eed-f:ctacacgacgctcttccgatctagttggagagagtatgttttctatacttgagatga(seq id no.83)eed-r: agacgtgtgctcttccgatctaactagatacaggattctcttgggtgatagtg(seq id no.84)
[0226]
bcl11a-f:ctacacgacgctcttccgatcttattttaatggaattgaaatgtgatatcctgtggtttatgatg(seq id no.85)
[0227]
bcl11a-r:agacgtgtgctcttccgatctccttcagtacttaaaaagtaagggcaatttccagaaa(seq id no.86)
[0228]
srd5a3-f:ctacacgacgctcttccgatcttgcctcgcttgttttccttttgcagat(seq id no.87)srd5a3-r: agacgtgtgctcttccgatctgcccttctgaggaggtgccg(seq id no.88)
[0229]
srd5a1-f:ctacacgacgctcttccgatctgaaattttacggtttattagccataatcatcttgcaa(seq id no.89)
[0230]
srd5a1-r:agacgtgtgctcttccgatctgatgacttacactgaaaacttgtagctaataactactacg(seq id no.90)
[0231]
dyrk1a-f:ctacacgacgctcttccgatctcttgtcacacacaatgaaactttgctgttcact(seq id no.91)
[0232]
dyrk1a-r:agacgtgtgctcttccgatctagtagctaacatttttcagtacttaactatatgccagctat(seq id no.92)
[0233]
dmd-f:ctacacgacgctcttccgatctaagcattaaatcttaagactacaagacattacttgaagg (seq id no.93)dmd-r:agacgtgtgctcttccgatctgacagagaagggtgtaaaagcttctagc (seq id no.94)
[0234]
runx1-f:ctacacgacgctcttccgatcttggttttcgctccgaaggtaaaagaa(seq id no.95)runx1
‑ꢀ
r:agacgtgtgctcttccgatctatatgcctcagtttgaattcctctcacaaacaag(seq id no.96)
[0235]
gfap-f：ctacacgacgctcttccgatcttactggggaaagcagtgcaggagca(seq id no.97)gfap-r: agacgtgtgctcttccgatctaccaccgcttcacagctgtgc(seq id no.98)
[0236]
补充表格3
[0237]
gene
[0238]
jak2:gtggcggcatgattttgtgcacggatggataaaagtacctgtgactcatgaaacaca ggaag
aatgtcttgggatggcagtgttagatatgatgagaatagccaaagaaaacgatcaaaccccactggccatctataactctatcaggtaattttcttttgcaaatccttacacataagtgtgagtagagattttatataattcgtatatattttctgtgtttacccatgccttttgattttgtaatactagttaagtactccttatctaaaatgcttgg(seqidno.99)
[0239]
hdac1:tcatgcccagcaagtgctgtgaaacttaataagcagcagacggacatcgctgtgaattgggctgggggcctgcaccatgcaaagaagtccgaggcatctggcttctgttacgtcaatgatatcgtcttggccatcctggaactgctaaagtatgcctgcctggccttgtctcttggaagagcaccttaggccaggttcccatttccctcttcccctgggcttgcctccctagtttgcttttcctaccgatgtgctggctaggatgtgctcggtgagtgtc(seqidno.100)
[0240]
eed:agttggagagagtatgttttctatacttgagatgaaatttactgtatttttattttcattaggttaccttgtatgaatgtcattcacaaggagaaatccggttgttgcaatcttacgtggatgctgatgtatcctttcctgggtttttaatatctttagtcaaaggaatttattttcaataagtgcaccaaaacttttataaattaatcatttccctaaagttattgttctttcttaaccgatttcttcactatcacccaagagaatcctgtatctagtt(seqidno.101)
[0241]
bcl11a:tattttaatggaattgaaatgtgatatcctgtggtttatgatgcacgttgtttgtagctgtagtgcttgattttgggtttctttcacagataaacttctgcactggaggggcctctcctcccctcgttctgcacatggagctctaatccccacgcctgggatgagtgcagaatatgccccgcagggtatttgtaagttgagccttatttcttctacaaatgtccatgtgtatagagatgagaatttctggaaattgcccttactttttaagtactgaagg(seqidno.102)
[0242]
srd5a3:tgcctcgcttgttttccttttgcagatatttttcccacttttatatcatctcagtgctgtggaatggcttcctgctttggtgccttactcaatctctgttcctgggagcaccttttccaagctggcttcatggtttgctcagaattctcggggcggcacagttccagggtaaggactccctgggcttatgacaacgctgcatcccgtttctgttgttcctgtctgcaagggagcagtttttggcattttgtctcctctctcggcacctcctcagaagggc(seqidno.103)
[0243]
srd5a1:
[0244]
gaaattttacggtttattagccataatcatcttgcaatttttttcctttaggttttggcttgtggttaacgggcatgttgataaacatccattcagatcatatcctaaggaatctcagaaaaccaggagatactggatacaaaataccaaggggtacgtacagaaagtgaagaatttctgtgaaagttgcttgccatggttcctggctattttagtgttgccagctctaagaagtagtagcgtagtagttattagctacaagttttcagtgtaagtcatc(seqidno.104)
[0245]
dyrk1a:
[0246]
cttgtcacacacaatgaaactttgctgttcactgtcagttataacttacatgaggtgacccatttccattcaagggttttagaagcacatcaaggacattctaaggatgattgacttacacaatgatctctgaacatgcctcctgccttctcctcactcttgagtatttgcttaggggagcagaaaaaatactggtagcattactgtaacattttaattctgctttatttaaaaaagacatagctggcatatagttaagtactgaaaaatgttagctact(seqidno.105)
[0247]
dmd:
[0248]
aagcattaaatcttaagactacaagacattacttgaaggtcaatgctctccttttcacaggctactcaaaaagagattgagaaacagaaggtgcacctgaagagtatcacagaggtaggagaggccttgaaaacagttttgg
gcaagaaggagacgttggtggaagataaactcagtcttct gaatagtaactggatagctgtcacctcccgagcagaagagtggttaaatcttttgttggta agagaaaaggctagaagcttttacacccttctctgtc(seq id no.106)
[0249]
runx1:
[0250]
tggttttcgctccgaaggtaaaagaaatcattgagtcccccgccttcagaagagggtgcattttca ggaggaagcgatggcttcagacagcatatttgagtcatttccttcgtacccacagtgctt catgagaggtgagtacatgctggtcttgtaatatctacttttgctcagctttgcctgtaatgaaatggcagcttgtttcacctcggtgc agagatgcctcggtgcctgccagttccctgtcttgtttgtgagaggaattcaaactgaggcatat(seq id no.107)
[0251]
gfap:
[0252]
tactggggaaagcagtgcaggagcagcggggcccctgtgtttcattcatggctgggctttgtgactgtgggcagcgagctcacctat tctgagcctgtgtccatataaaggaggagttggaagcggagaaggttgatgtccatgagggagattggattctggggtgaagaaa gtgagggaaagagcaggcaggtctgggcgcaaagcacaggtgactgcctgccaccagcttgtgacccccatcaagttactttga cttgcacagctgtgaagcggtggt(seq id no.108)
[0253]
补充序列1
[0254]
pdonor 5.1
[0255]
acggatcgggagatctaccggtacgcgttgacgtgtcttcggatccggcttactaaaagccagataacagtatgcgtatttgcgcgc tgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtatgctatgaagcagcgtattacagtgac agttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagc ccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctct tttgctgacgagaacaggggctggtgaaatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtac agagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaa ctttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcgggga agaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggctcc cttatacacagccagtctgcaggtcgacgaagacccactcgcggccgcttggggttgcgccttttccaaggcagccctgggtttgcg cagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgca gcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggtt cgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggc agcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaaggggcgg tgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcac gtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatccgccaccatgaccgagtacaagcccacg gtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgcca caccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaa ggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgag atcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggc ccaaggagcccgcgtggttcctggccaccgt
cggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctcc ccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcgg ctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgccgctag cggtgctactaacttcagcctgctgaagcaggctggagacgtggaggagaaccctggacctggtagtggaacgcgtatggtgtct aagggcgaagagctgattaaggagaacatgcacatgaagctgtacatggagggcaccgtggacaaccatcacttcaagtgcac atccgagggcgaaggcaagccctacgagggcacccagaccatgagaatcaaggtggtcgagggcggccctctccccttcgcct tcgacatcctggctactagcttcctctacggcagcaagaccttcatcaaccacacccagggcatccccgacttcttcaagcagtcctt ccctgagggcttcacatgggagagagtcaccacatacgaagatgggggcgtgctgaccgctacccaggacaccagcctccag gacggctgcctcatctacaacgtcaagatcagaggggtgaacttcacatccaacggccctgtgatgcagaagaaaacactcggc tgggaggccttcaccgagacgctgtaccccgctgacggcggcctggaaggcagaaacgacatggccctgaagctcgtgggcg ggagccatctgatcgcaaacgccaagaccacatatagatccaagaaacccgctaagaacctcaagatgcctggcatctactatg tggactacagactggaaagaatcaaggaggccaacaacgaaacctacgtcgagcagcacgaggtggcagtggccagatact gcgacctccctagcaaactggggcacaagcttaatccaaagaaaaagcggaaagtgtgagtcgacctcgagggggggcccg gtacctttaagaccaatgacttacaaggcagctgtagatcttagccacatgtcatcttattattcagtatttataacttgcaaagaaatga atatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcat ttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggaattcacatgtgagcaaaaggccagcaaaa ggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctca agtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgac cctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtg taggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagt ccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgcta cagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcgg aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaa aaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatg agattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctg acagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagat aactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatca gcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccg ggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttgg tatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtc ctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgt aagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaa tacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatc ttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagc aaaaacaggaaggcaaaatgc
cgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatat tattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcaca tttccccgaaaagtgccacctgacgtc(seq id no.109)
[0256]
plh-aaaa
[0257]
ttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgc cgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgctt cctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagta ccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcag ggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccg cgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccag ggggatccaccggatcaagcttgccagctggggcgccctctggtaaggttgggaagccctgcagatctgatcccggggggcaat gagatatgaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgca gctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatgg tttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaatttagcgagag cctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagatctgcctgaaaccgaactgcccgctgttctgcagccggt cgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtc aatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgc gtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggct ccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgagg tcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcag gatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagct tgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcgg ccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaatag agtagatgccgaccgggatctatcgataccgtcgacctcgagggggggcccggtacctttaagaccaatgacttacaaggcagct gtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagaaaagatctgctttttgcttgta ctgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgcc ttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctcta gcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagc ttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactca tcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctcc gccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctttttt ggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactggg aaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccga tcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggtt acgcgcagcgtgaccgctacacttgcca
gcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttc cccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgat ggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaac tggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgattta acaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttg tttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatg agtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaag atgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaag aacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaatta tgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttt tttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgac accacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaata gactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagcc ggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggag tcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttt actcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccc ttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgct gcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggctt cagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacata cctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccgg ataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagata cctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcgg aacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagc gtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagc cgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctca ctcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaac agctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttat gcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgatt ggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccg cattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctgg ctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgt
gtgcccgtctgttgtgtgactctggtaa ctagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaa ccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgc caaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcga tgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaac gattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacagg atcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaa gctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggagg aggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaa ggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaa gcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctg agggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaa agatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttg gagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaata cactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtgga attggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtac tttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggccc gaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgatcac gagactagcctcgagcggccgcccccttcaccgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgc agttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggac gaaacaccggtgtcttcgaggcttactaaaagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactg atatgtatacccgaagtatgtcaaaaagaggtgtgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgct caaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaa agcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggactggtga aatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggc gacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatg aaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcga aaatgacatcaaaaacgccattaacctgatgttctggggaatataaaccctgcaggacgaagaccctttttttgaattctcgacctcg agacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacat aatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcaga gatccactttggccgcggctcgaggggg(seq id no.110)
[0258]
plh-sgrna3
[0259]
ttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgc cgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgc
cgctacccttgtgggccccccggcgacgctt cctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagta ccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcag ggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccg cgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccag ggggatccaccggatcaagcttgccagctggggcgccctctggtaaggttgggaagccctgcagatctgatcccggggggcaat gagatatgaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgca gctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatgg tttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaatttagcgagag cctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagatctgcctgaaaccgaactgcccgctgttctgcagccggt cgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtc aatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgc gtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggct ccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgagg tcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcag gatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagct tgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcgg ccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaatag agtagatgccgaccgggatctatcgataccgtcgacctcgagggggggcccggtacctttaagaccaatgacttacaaggcagct gtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagaaaagatctgctttttgcttgta ctgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgcc ttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctcta gcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagc ttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactca tcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctcc gccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctttttt ggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactggg aaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccga tcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggtt acgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttc cccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgat ggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaac tggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgattta acaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttg tttatttttctaaatacattcaaatat
gtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatg agtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaag atgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaag aacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaatta tgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttt tttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgac accacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaata gactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagcc ggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggag tcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttt actcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccc ttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgct gcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggctt cagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacata cctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccgg ataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagata cctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcgg aacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagc gtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagc cgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctca ctcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaac agctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttat gcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgatt ggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccg cattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctgg ctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaa ctagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaa ccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgc caaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcga tgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaac gattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacagg atcaga
agaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaa gctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggagg aggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaa ggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaa gcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctg agggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaa agatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttg gagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaata cactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtgga attggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtac tttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggccc gaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgatcac gagactagcctcgagcggccgcccccttcaccgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgc agttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggac gaaacaccggtgtcttcgaggcttactaaaagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactg atatgtatacccgaagtatgtcaaaaagaggtgtgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgct caaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaa agcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggactggtga aatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggc gacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatg aaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcga aaatgacatcaaaaacgccattaacctgatgttctggggaatataaaccctgcaggacgaagacccgttttagagctagaaatag caagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgaattctcgacctcgagacaaatgg cagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaaca gacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccactttg gccgcggctcgaggggg(seq id no.111)
[0260]
pcmv-fkbp-mcp
[0261]
acggatcgggagatctaccggtacgcgttgacagagacgggatccgacattgattattgactagttattaatagtaatcaattacgg ggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgaccccc gcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcat tatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggca gtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtt
tgttttggcacc aaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtcta tataagcagagctggtttagtgaaccgtcagatccgctagagatccgcggccgctaatacgactcactatagggagagccgccac catgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgcactaca ccgggatgcttgaagatggaaagaaatttgattcctctcgggacagaaacaagccctttaagtttatgctaggcaagcaggaggtg atccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactatatctccagattatgcctatggtgc cactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaaaacggacagccgac ggaagcgagttcgagtcaccaaagaagaagcggaaagtcatggcttctaactttactcagttcgttctcgtcgacaatggcggaac tggcgacgtgactgttgccccaagcaacttcgctaacgggatcgctgaatggatcagctctaactcgcgttcacaggcttacaaagt aacctgtagcgttcgtcagagctctgcgcagaatcgcaaatacaccatcaaagtcgaggtgcctaaaggcgcctggcgttcgtact taaatatggaactaaccattccaattttcgccacgaattccgactgcgagcttattgttaaggcaatgcaaggtctcctaaaagatgg aaacccgattccctcagcaatcgcagcaaactccggcatctacgagggcagaggaagtcttctaacatgcggtgacgtggagga gaatcccggccctgctagcggtactagcatggcagaaatcggtactggctttccattcgacccccattatgtggaagtcctgggcga gcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgc aacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgg gttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactgg ggctccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccga cctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatc agaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcc tgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggt cgaagaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccggcc gaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctgcaagaagacaa cccggacctgatcggcagcgagatcgcgcgctggctgtcgacgctggagatttccggctaagtttaaacccgctgatcagcctcga ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaat aaaatgagaaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggagga ttgggaagacaatagcaggcatgctggggatgcggtgggctctatgggcggccgcgaattcacatgtgagcaaaaggccagca aaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttc cgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttc ggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtctt gagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcgg tgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacct tcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgca gaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggt catgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatat
atgagtaaacttg gtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgt agataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagattt atcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgtt gccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtc gtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctcctt cggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgcca tccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggc gtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaa ggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctggg tgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttccttttt caatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccg cgcacatttccccgaaaagtgccacctgacgtc(seq id no.112)
[0262]
pcmv-pyl1-mcp
[0263]
acggatcgggagatctaccggtacgcgttgacagagacgggatccgacattgattattgactagttattaatagtaatcaattacgg ggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgaccccc gcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcat tatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggca gtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcacc aaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtcta tataagcagagctggtttagtgaaccgtcagatccgctagagatccgcggccgctaatacgactcactatagggagagccgccac cggtgggggcgcgccaactcaagacgaattcacccaactctcccaatcaatcgccgagttccacacgtaccaactcggtaacgg ccgttgctcatctctcctagctcagcgaatccacgcgccgccggaaacagtatggtccgtggtgagacgtttcgataggccacagat ttacaaacacttcatcaaaagctgtaacgtgagtgaagatttcgagatgcgagtgggatgcacgcgcgacgtgaacgtgataagt ggattaccggcgaatacgtctcgagagagattagatctgttggacgatgatcggagagtgactgggtttagtataaccggtggtgaa cataggctgaggaattataaatcggttacgacggttcatagatttgagaaagaagaagaagaagaaaggatctggaccgttgtttt ggaatcttatgttgttgatgtaccggaaggtaattcggaggaagatacgagattgtttgctgatacggttattagattgaatcttcagaa acttgcttcgatcactgaagctatgaacaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtc atggcttctaactttactcagttcgttctcgtcgacaatggcggaactggcgacgtgactgttgccccaagcaacttcgctaacgggat cgctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaata caccatcaaagtcgaggtgcctaaaggcgcctggcgttcgtacttaaatatggaactaaccattccaattttcgccacgaattccga ctgcgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatct acgagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctgctagcggtactagcatggcagaaatc ggtactggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgt
cgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtcgacgctggagatttccggctaagtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgagaaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggcggccgcgaattcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagaaccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc(seqidno.113)
[0264]
pcmv-frb-pe2-t2a-gfp
[0265]
gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacg gtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatag ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcc ccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaa gtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccatt gacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagagatcc gcggccgctaatacgactcactatagggagagccgccaccatgatcctctggcatgagatgtggcatgaaggcctggaagaggc atctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggacccca gactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatca gggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctcaaagaaacggacagccgacgga agcgagttcgagtcaccaaagaagaagcggaaagtcgacaagaagtacagcatcggcctggacatcggcaccaactctgtgg gctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaa gaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaa gatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttcc acagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtg gcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctga tctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtgga caagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaagg ccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctg ttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagc tgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgcc aagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatg atcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaag agattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaa gcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggac cttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattc ctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacag cagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccg cccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacg agtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgag cagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaaga aaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaa attatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgt
gctgaccctgacactgtttgagg acagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggag atacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcc tgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaag cccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctg cagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagag agaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgg gcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgg gcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcctcagagctttctga aggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagag gtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaa ggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaa gcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatca ccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccac gacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactac aaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaa catcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaac cggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaa gaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaa ggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaaggg caagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcg actttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaa cggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcct gtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagc actacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtc cgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagc ccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgat ccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggaggatctagcggaggatc ctctggcagcgagacaccaggaacaagcgagtcagcaacaccagagagcagtggcggcagcagcggcggcagcagcacc ctaaatatagaagatgagtatcggctacatgagacctcaaaagagccagatgtttctctagggtccacatggctgtctgattttcctca ggcctgggcggaaaccgggggcatgggactggcagttcgccaagctcctctgatcatacctctgaaagcaacctctacccccgtg tccataaaacaataccccatgtcacaagaagccagactggggatcaagccccacatacagagactgttggaccagggaatact ggtaccctgccagtccccctggaacacgcccctgctacccgttaagaaaccagggactaatgattataggcctgtccaggatctga gagaagtcaacaagcgggtggaagacatccaccccaccgtgcccaacccttacaacctcttgagcgggctcccaccgtcccac cagtggtacactgtgcttgatttaaaggatgcctttttctgcctgagactccaccccaccagtcagcctctcttcgcctttgagtggaga gatccagagatgggaatctcaggacaattgacctggaccagactcccacagggtttcaaaaacagt
agaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttg ctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacagga ctataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgccttt ctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtg cacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgcc actggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactac ggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggca aacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttga tcttttctacggggtctgacactcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctag atccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgagg cacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatc tggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaaggg ccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagtt aatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaac gatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggc cgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactc aaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagca gaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaa cccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgca aaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctca tgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcg acggatcgggagatcgatctcccgatcccctagggtctactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctc cctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaa gaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgtt(seq id no.114)
[0266]
pcmv-abi-pe2-t2a-gfp
[0267]
gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacg gtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatag ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgcc ccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaa gtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccatt gacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagagatcc gcggccgctaatacgactcactatagggagagccgccaccatgacgcgtgtgcctttgtatggttttacttcgatttgtggaagaaga cctgagatggaagctgctgtttcgactataccaagattcc
ttcaatcttcctctggttcgatgttagatggtcggtttgatcctcaatccgc cgctcatttcttcggtgtttacgacggccatggcggttctcaggtagcgaactattgtagagagaggatgcatttggctttggcggagg agatagctaaggagaaaccgatgctctgcgatggtgatacgtggctggagaagtggaagaaagctcttttcaactcgttcctgaga gttgactcggagattgagtcagttgcgccggagacggttgggtcaacgtcggtggttgccgttgttttcccgtctcacatcttcgtcgcta actgcggtgactctagagccgttctttgccgcggcaaaactgcacttccattatccgttgaccataaaccggatagagaagatgaag ctgcgaggattgaagccgcaggagggaaagtgattcagtggaatggagctcgtgttttcggtgttctcgccatgtcgagatccattg gcgatagatacttgaaaccatccatcattcctgatccggaagtgacggctgtgaagagagtaaaagaagatgattgtctgattttgg cgagtgacggggtttgggatgtaatgacggatgaagaagcgtgtgagatggcaaggaagcggattctcttgtggcacaagaaaa acgcggtggctggggatgcatcgttgctcgcggatgagcggagaaaggaagggaaagatcctgcggcgatgtccgcggctgag tatttgtcaaagctggcgatacagagaggaagcaaagacaacataagtgtggtggtggttgatttgaagaaacggacagccgac ggaagcgagttcgagtcaccaaagaagaagcggaaagtcgacaagaagtacagcatcggcctggacatcggcaccaactct gtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagca tcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaa gaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttc ttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgag gtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggc tgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgt ggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgcca aggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatgg cctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactg cagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggc cgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcct ctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtac aaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttc atcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcag cggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttac ccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccagggga aacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggc gcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctg ctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagc ggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactact tcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctg ctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtt tgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcgg cggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctg gatttcctgaagtccgacggcttcgccaa
cagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatcc agaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaaggg catcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatgg ccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaag agctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgca gaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggacgctatcgtgcctcagag ctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctcc gaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatct gaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagat cacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaa gtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccac gcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggc gactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttcta cagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacgg cgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgt gaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccag aaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtgga aaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaat cccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagct ggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtg aacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagc acaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaag tgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatct gggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgcca ccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggaggatctagcg gaggatcctctggcagcgagacaccaggaacaagcgagtcagcaacaccagagagcagtggcggcagcagcggcggcag cagcaccctaaatatagaagatgagtatcggctacatgagacctcaaaagagccagatgtttctctagggtccacatggctgtctg attttcctcaggcctgggcggaaaccgggggcatgggactggcagttcgccaagctcctctgatcatacctctgaaagcaacctct acccccgtgtccataaaacaataccccatgtcacaagaagccagactggggatcaagccccacatacagagactgttggacca gggaatactggtaccctgccagtccccctggaacacgcccctgctacccgttaagaaaccagggactaatgattataggcctgtcc aggatctgagagaagtcaacaagcgggtggaagacatccaccccaccgtgcccaacccttacaacctcttgagcgggctccca ccgtcccaccagtggtacactgtgcttgatttaaaggatgcctttttctgcctgagactccaccccaccagtcagcctctcttcgcctttg agtggagagatccagagatgggaatctcaggacaattgacctggaccagactcccacagggtttcaaaaacagtcccaccctgtt taatgaggcactgcacagagacctagcagacttccggatccagcacccagacttgatcctgctacagtacgtggatgacttactgc tggccgccacttctgagctagactgccaacaaggtactcgggccctgttacaaaccctagggaacctcgggtatcgggcctcggc caagaaagcccaaatttgccagaaacagg
tcaagtatctggggtatcttctaaaagagggtcagagatggctgactgaggccag aaaagagactgtgatggggcagcctactccgaagacccctcgacaactaagggagttcctagggaaggcaggcttctgtcgcct cttcatccctgggtttgcagaaatggcagcccccctgtaccctctcaccaaaccggggactctgtttaattggggcccagaccaaca aaaggcctatcaagaaatcaagcaagctcttctaactgccccagccctggggttgccagatttgactaagccctttgaactctttgtc gacgagaagcagggctacgccaaaggtgtcctaacgcaaaaactgggaccttggcgtcggccggtggcctacctgtccaaaaa gctagacccagtagcagctgggtggcccccttgcctacggatggtagcagccattgccgtactgacaaaggatgcaggcaagct aaccatgggacagccactagtcattctggccccccatgcagtagaggcactagtcaaacaaccccccgaccgctggctttccaac gcccggatgactcactatcaggccttgcttttggacacggaccgggtccagttcggaccggtggtagccctgaacccggctacgct gctcccactgcctgaggaagggctgcaacacaactgccttgatatcctggccgaagcccacggaacccgacccgacctaacgg accagccgctcccagacgccgaccacacctggtacacggatggaagcagtctcttacaagagggacagcgtaaggcgggag ctgcggtgaccaccgagaccgaggtaatctgggctaaagccctgccagccgggacatccgctcagcgggctgaactgatagca ctcacccaggccctaaagatggcagaaggtaagaagctaaatgtttatactgatagccgttatgcttttgctactgcccatatccatg gagaaatatacagaaggcgtgggtggctcacatcagaaggcaaagagatcaaaaataaagacgagatcttggccctactaaa agccctctttctgcccaaaagacttagcataatccattgtccaggacatcaaaagggacacagcgccgaggctagaggcaaccg gatggctgaccaagcggcccgaaaggcagccatcacagagactccagacacctctaccctcctcatagaaaattcatcaccctc tggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtcgagggcagaggaagtcttct aacatgcggtgacgtggaggagaatcccggccctgctagcggtactagcatgcgtaaaggcgaagagctgttcactggtgtcgtc cctattctggtggaactggatggtgatgtcaacggtcataagttttccgtgcgtggcgagggtgaaggtgacgcaactaatggtaaa ctgacgctgaagttcatctgtactactggtaaactgccggtaccttggccgactctggtaacgacgctgacttatggtgttcagtgctttg ctcgttatccggaccatatgaagcagcatgacttcttcaagtccgccatgccggaaggctatgtgcaggaacgcacgatttcctttaa ggatgacggcacgtacaaaacgcgtgcggaagtgaaatttgaaggcgataccctggtaaaccgcattgagctgaaaggcattg actttaaagaagacggcaatatcctgggccataagctggaatacaattttaacagccacaatgtttacatcaccgccgataaacaa aaaaatggcattaaagcgaattttaaaattcgccacaacgtggaggatggcagcgtgcagctggctgatcactaccagcaaaac actccaatcggtgatggtcctgttctgctgccagacaatcactatctgagcacgcaaagcgttctgtctaaagatccgaacgagaaa cgcgatcatatggttctgctggagttcgtaaccgcagcgggcatcacgcatggtatggatgaactgtacaaataagtttaaacccgc tgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactccca ctgtcctttcctaataaaatgagaaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagc aagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctg gggctcgataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattcca cacaacatacgagccggaagcataaagtgtaaagcctagggtgcctaatgagtgagctaactcacattaattgcgttgcgctcact gcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggc gctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacg gttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaagg ccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaaccc gacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctc
ctgttccgaccctgccgcttaccggatacctg tccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgg gctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgact tatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcct aactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgat ccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaaga tcctttgatcttttctacggggtctgacactcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttc acctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatca gtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggct taccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccg gaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagtt cgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggt tcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagta agttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggt gagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgcc acatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagt tcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaa atgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggt tattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac ctgacgtcgacggatcgggagatcgatctcccgatcccctagggtctactctcagtacaatctgctctgatgccgcatagttaagcca gtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaa ttgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgtt(seq id no.115)
[0268]
补充序列2
[0269]
mcp
[0270]
atggcttctaactttactcagttcgttctcgtcgacaatggcggaactggcgacgtgactgttgccccaagcaacttcgctaacgggat cgctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaata caccatcaaagtcgaggtgcctaaaggcgcctggcgttcgtacttaaatatggaactaaccattccaattttcgccacgaattccga ctgcgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatct ac(seq id no.116)
[0271]
tdmcp
[0272]
gcttctaactttactcagttcgttctcgtcgacaatggcggaactggcgacgtgactgtcgccccaagcaacttcgctaacgggatcg ctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaataca ccatcaaagtcgaggtgcctaaaggcgcctggcgttcgtacttaaatatggaactaaccattccaattttcgccacgaattccgactg cgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatctacg ccatggccagcaacttcac
ccagttcgtgctggtggacaacggcggcaccggcgacgtgaccgtggcccccagcaacttcgcca acggcatcgccgagtggatcagcagcaacagcagaagccaggcctacaaggtgacctgcagcgtgagacagagcagcgcc cagaacagaaagtacaccatcaaggtggaggtgcccaagggcgcctggagaagctacctgaacatggagctgaccatcccca tcttcgccaccaacagcgactgcgagctgatcgtgaaggccatgcagggcctgctgaaggacggcaaccccatccccagcgcc atcgccgccaacagcggcatctacgcc(seq id no.117)
[0273]
com
[0274]
ggtagtatgaaatcaattcgctgtaaaaactgcaacaaactgttatttaaggcggatagttttgatcacattgaaatcaggtgtccgcg ttgcaaacgtcacatcataatgctgaatgcctgcgagcatcccacggagaaacattgtgggaaaagagaaaaaatcacgcattct gacgaaaccgtgcgttatggctccactagtgggcacaagcttaatggcggtggcggagggatggatgctaagtcactaactgcct ggtcc(seq id no.118)
[0275]
csy4h29a
[0276]
atggaccactacctcgacattcgcttgcgaccggacccggaatttcccccggcgcaactcatgagcgtgctcttcggcaagctcgc ccaggccctggtggcacagggcggggacaggatcggcgtgagcttccccgacctcgacgaaagccgctcccggctgggcgag cgcctgcgcattcatgcctcggcggacgaccttcgtgccctgctcgcccggccctggctggaagggttgcgggaccatctgcaattc ggagaaccggcagtcgtgcctcaccccacaccgtaccgtcaggtcagtcgggttcaggcgaaaagcaatccggaacgcctgcg gcggcggctcatgcgccggcacgatctgagtgaggaggaggctcggaaacgcattcccgatacggtcgcgagagccttggacc tgcccttcgtcacgctacgcagccagagcaccggacagcacttccgtctcttcatccgccacgggccgttgcaggtgacggcaga ggaaggaggattcacctgttacgggttgagcaaaggaggtttcgttccctggttc(seq id no.119)
[0277]
pcp
[0278]
atgggttccaaaaccatcgttctttcggtcggcgaggctactcgcactctgactgagatccagtccaccgcagaccgtcagatcttc gaagagaaggtcgggcctctggtgggtcggctgcgcctcacggcttcgctccgtcaaaacggagccaagaccgcgtatcgcgtc aacctaaaactggatcaggcggacgtcgttgattccggacttccgaaagtgcgctacactcaggtatggtcgcacgacgtgacaat cgttgcgaatagcaccgaggcctcgcgcaaatcgttgtacgatttgaccaagtccctcgtcgcgacctcgcaggtcgaagatcttgt cgtcaaccttgtgccgctgggccgt(seq id no.120)
[0279]
tdpcp
[0280]
tccaaaactatcgttctgagcgtaggtgaagcgaccagaacgttaactgaaattcaatctacagcagacaggcaaatatttgaaga gaaagtcggtccattagtagggaggttaagattgacagctagcttaagacaaaatggagctaaaactgcatacagggtcaatttaa aattggatcaagcagatgtcgttgatagcggtctacctaaggttagatacacgcaagtgtggtcacacgatgtcactattgtcgcgaa ttctaccgaagctagtagaaaatcattgtatgatttaacaaaatccttggtggctacctctcaggtggaagatcttgttgtgaatttagttc cattaggtagggcggacccgctagcctccaaaaccatcgttctttcggtcggcgaggctactcgcactctgactgagatccagtcca ccgcagaccgtcagatcttcgaagagaaggtcgggcctctggtgggtcggctgcgcctcacggcttcgctccgtcaaaacggagc caagaccgcgtatcgcgtcaacctaaaactggatcaggcggacgtcgttgattccggacttccgaaagtgcgctacactcaggtat ggtcgcacgacgtgacaatcgttgcgaatagcaccgaggcctcgcgcaaatcgttgtacgatttgaccaagtccctcgtcgcgacc tcgcaggtcgaagatcttgtcgtcaaccttgtgccgctgggccgt(seq id no.121)

技术特征：
1.一种基因先导编辑系统，包含载体，所述载体含有编码sgrna的基因、编码分离的先导rna的基因以及编码融合蛋白的基因，其特征在于，所述分离的先导rna包含逆转录模板rtt、主要结合位点pbs、rna茎环结构。2.如权利要求1所述的系统，其特征在于，所述融合蛋白为rna结合蛋白rbp、cas9切口酶或其变体、逆转录酶的融合蛋白；或，所述融合蛋白包括：包含rna结合蛋白rbp与第一诱导结合分子的第一融合蛋白，以及包含cas9切口酶或其变体、逆转录酶、第二诱导结合分子的第二融合蛋白片段。3.如权利要求1所述的系统，其特征在于，所述rna茎环结构选自以下至少一项：1)序列分别如seq id no.1～5所示的ms2、pp7、combs、csy4bs、boxb；2)在1)的5
′
端和/或3
′
端添加一个或几个核苷酸得到的rna序列；3)与1)或2)的rna序列具有85％以上的同一性的rna序列。4.根据权利要求1所述的系统，其特征在于，选自以下至少一项：1)所述rna茎环结构连接于所述主要结合位点pbs的3’端，和/或逆转录模板rtt的5’端；2)所述分离的先导rna从5’端至3’端依次为逆转录模板rtt、主要结合位点pbs和rna茎环结构；3)所述分离的先导rna从5’端至3’端依次为rna茎环结构、逆转录模板rtt、主要结合位点pbs；4)所述分离的先导rna为线性或环形；5)所述rbp为特异性结合rna茎环结构的蛋白；所述rbp为一个或多个；6)所述载体为一个或多个；7)所述编码sgrna的基因、编码分离的先导rna的基因以及编码融合蛋白的基因为一个或多个；8)所述编码sgrna的基因、编码分离的先导rna的基因以及编码融合蛋白的基因位于同一载体或不同载体上；9)所述第一诱导结合分子、第二诱导结合分子为适于通过诱导分子进行结合的分子；10)所述第一诱导结合分子位于rbp的n端或c端；11)所述第二融合蛋白片段由n端至c端依次包括第二诱导结合分子、cas9或其变体、逆转录酶；或，所述第二融合蛋白片段由n端至c端依次包括cas9或其变体、逆转录酶、第二诱导结合分子。5.根据权利要求4所述的系统，其特征在于，选自以下任一项：1)所述rbp选自tdmcp、tdpcp、com、csy4h29a、n22p中的任一种；2)所述cas9切口酶为h840a；3)所述逆转录酶为mmlv；3)所述第一诱导分子、第二诱导分子分别独立地选自两对蛋白fkbp和frb或者abi和pyl1中的一种；4)所述系统还含有筛选标记蛋白。6.一种分离的先导rna，其特征在于，所述分离的先导为权利要求1～5任一项所述的系统中所述的分离的先导rna。
7.一种融合蛋白，其特征在于，所述融合蛋白包含rna结合蛋白rbp与第一诱导结合分子，或者，所述融合蛋白包含cas9切口酶或其变体、逆转录酶和第二诱导结合分子。8.根据权利要求7所述的融合蛋白，其特征在于，选自以下至少任一项：1)所述第一诱导结合分子、第二诱导结合分子为适于通过诱导分子进行结合的分子；2)所述第一诱导分子、第二诱导分子分别独立地选自两对蛋白fkbp和frb或者abi和pyl1中的一対；3)所述cas9切口酶为h840a；4)所述逆转录酶为mmlv。9.一种核酸，其特征在于，所述核酸编码权利要求6所述的分离的先导rna和/或权利要求7或8所述的融合蛋白。10.一种构建体，所述构建体含有如权利要求9所述的核酸。11.一种宿主细胞，其特征在于，所述宿主细胞包含如权利要求10所述的构建体或基因组中整合有外源的如权利要求9所述的核酸。12.根据权利要求1-5任一项所述的系统或权利要求6所述的分离的先导rna或权利要求7或8所述的融合蛋白或权利要求9所述的核酸或权利要求10所述的构建体或权利要求11所述的宿主细胞的以下至少一项应用：1)用于生物体或生物细胞基因组序列的编辑；2)用于制备生物体或生物细胞基因组序列的编辑的产品；3)用于提高生物体或生物细胞基因组序列的编辑效率；4)用于制备提高生物体或生物细胞基因组序列的编辑效率的产品。13.根据权利要求12所述的应用，其特征在于，用于dna编辑或rna编辑；优选地，用于dna编辑；进一步优选地，用于runx1_+5位点的编辑；更进一步优选地，用于runx1_+5g
·
c到t
·
a的编辑。14.一种基因编辑的方法，包括将权利要求1-5任一项所述的系统或权利要求6所述的分离的先导rna或权利要求7或8所述的融合蛋白或权利要求9所述的核酸或权利要求10所述的构建体或权利要求11所述的宿主细胞导入待编辑体中实现基因的编辑。

技术总结
本发明属于生物医药技术领域，公开了一种基因先导编辑系统，包含载体，所述载体含有编码sgRNA的基因、编码分离的先导RNA(pRNA)的基因以及编码融合蛋白的基因，所述分离的先导RNA包含逆转录模板RTT、主要结合位点PBS、RNA茎环结构。本发明通过将3


技术研发人员：马涵慧 冯颖
受保护的技术使用者：上海科技大学
技术研发日：2022.01.30
技术公布日：2023/8/9