CN114058607A - 一种用于c到u碱基编辑的融合蛋白及其制备方法和应用 - Google Patents

一种用于c到u碱基编辑的融合蛋白及其制备方法和应用 Download PDF

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CN114058607A
CN114058607A CN202010764514.XA CN202010764514A CN114058607A CN 114058607 A CN114058607 A CN 114058607A CN 202010764514 A CN202010764514 A CN 202010764514A CN 114058607 A CN114058607 A CN 114058607A
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池天
黄欣欣
吕君君
毛少帅
李永芹
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Abstract

本发明提供了一种融合蛋白，其包括APOBEC 3A片段和失活的dCas13片段，所述失活的dCas13片段包括dCas13Rx片段和/或dPspCas13b片段。本发明还提供了该融合蛋白的制备方法和应用。本发明进一步提供了一种用于检测所述融合蛋白编辑效率的荧光报告系统。本发明的融合蛋白用于碱基编辑时并不会破坏任何腺嘌呤，没有A到I的脱靶，并且该系统更倾向于编辑UC，也能编辑CC，对内源性转录本的编辑中UC序列的编辑效率较高。此外特异性也显著提高。将本发明的融合蛋白用于编辑编辑，不仅能给与C到U的碱基编辑提供了新的选择，而且也跟目前仅有的RNA的C到U的编辑编辑器RESCUE‑S互补。

Description

一种用于C到U碱基编辑的融合蛋白及其制备方法和应用
技术领域
本发明涉及生物技术领域，特别是涉及一种用于C到U的碱基编辑的融合蛋白及其制备方法和应用，本发明还涉及一种碱基编辑器以及碱基编辑方法。
背景技术
可编程的RNA编辑，包括A>I(A到I)和C>U(C到U)的编辑，是对基础研究和临床治疗中DNA碱基编辑(又称为基因编辑)的必要补充(Rees和Liu,2018)(Vogel和 stst,2019)。经过前期的努力，已经开发出许多版本的A>I RNA编辑器，所有这些编辑器都利用了人类RNA腺嘌呤脱氨酶ADAR蛋白(Cox et al.,2017；Katrekar et al.,2019； Montiel-Gonzalezet al.,2013；Montiel-Gonzalez et al.,2016；Rauch et al.,2019；Sinnamon et al.,2017；Vallecillo-Viejo et al.,2018；Vogel et al.,2018)。值得注意的是，C>U编辑器直到最近才出现。具体来说，新出现的C>U编辑器是将点突变和蛋白进化相结合，Zhang和同事成功地将人类ADAR2脱氨酶域转化为双功能酶，能够同时脱氨腺嘌呤和胞嘧啶，将修饰后的酶融合到失活的靶向RNA的CRISPR-Cas13(dCas13)蛋白上，形成可编程的C>U和A>I双功能编辑器RESCUE(Abudayyeh et al.,2019)。虽然RESCUE可以在相应合适的引导RNA存在的情况下，选择性的将胞嘧啶脱氨，但它同时造成大量腺嘌呤脱氨。在RESCUE体系中，将点突变(S375A)引入到ADAR2蛋白的脱氨酶区域，可以显著抑制脱靶，这是一个高特异性的RESCUE版本，称之为RESCUE-S(Abudayyeh et al., 2019)。然而，RESCUE-S中仍然存在一些A>I和C>U脱靶，即，除对靶位点脱氨外，还会对C和A有非特异性脱氨。此外，对已发表数据(Abudayyeh et al.,2019，见图19) 的重新分析表明，RESCUE在对内源性转录本的编辑中，对靶位点C前面的碱基有偏好性，更倾向于编辑AC和UC，G/CC(CC和GC)序列编辑效率较低，而因为RESCUE- S活性通常不如RESCUE，这种偏好现象在RESCUE-S中更为严重(图20)。事实上， RESCUE-S对内源性转录本中G/CC序列的编辑效率通常低于2％，这使得它实际上不实用。最后，虽然RESCUE-S可以编辑AC和UC，但UC编辑的效率较低，甚至在靶位点 UC的编辑率低于4％(图20)。因此，开发出类似A>I一样高效的C>U RNA碱基编辑器是很有必要的。
发明内容
为了克服现有技术中的RNA编辑器在选择性的将胞嘧啶脱氨的同时会造成大量腺嘌呤脱氨、存在A>I和C>U脱靶现象、对内源性转录本的编辑中G/CC序列编辑效率较低、实用性低、UC编辑的效率较低等缺陷，提供了一种用于C到U的碱基编辑的融合蛋白及其制备方法和应用，还提供了一种碱基编辑器以及碱基编辑方法。本发明进一步提供了一种用于检测所述融合蛋白编辑效率的荧光报告系统。
RESCUE的脱靶率太高，将RESCUE升级成RESCUE-S之后在某些靶点编辑效率变低，编辑效率有所下降。本发明人经过大量实验，尝试将各种APOBEC蛋白融合至Cas13，但是遇到各种困难，比如APOBEC1在DNA编辑中是可以的，但换上了RNA结合蛋白之后，仍然无法定点编辑，即使尝试加了Mooring还是不能定点编辑。本发明人尝试用 APOBEC 3A的策略让与APOBEC 3A(简称为A3A)识别结构很像的APOBEC 3G(简称为A3G)能扩宽C>U的编辑框(A3A只能编辑C前面是U的，而A3G据报道可以编辑C前面是C的)，最后也是失败的。目前在DNA上实现A>I与C>U的碱基编辑也已有众多报道，但是在张锋(Abudayyeh et al.,2019)还没报道进化出双功能RNA编辑器 (RESCUE-S)之前，市场上只存在A>I的RNA编辑，而且陆续发的文章也是跟A>I的编辑相关。本发明的发明人经过大量探索性研究，发现了一种融合蛋白，其是将APOBEC 3A蛋白融合到失活的dCas13蛋白，构成一种新的C到U的RNA编辑器(CURE)，能够实现在RNA上仅仅有C>U的碱基编辑，且具有更高的编辑效率和特异性。此外，本发明人在实验过程中用到的初步筛选编辑器的荧光报告系统，前期背景较高，即GFP两部分的蛋白自己跟自己相结合发光造成了背景，因此还进一步改进了GFP荧光报告系统，使其更加适用于检测本发明的融合蛋白。
为了解决上述技术问题，本发明第一方面提供了一种融合蛋白，其包括APOBEC 3A片段和失活的dCas13片段，所述失活的dCas13片段包括dCas13Rx(Ruminococcusflavefaciensstrain XPD3002)片段和/或dPspCas13b(源自普氏杆菌(Prevotella sp.)P5-125 的Cas13b)片段。
本申请中，所述的失活的dCas13片段一般是保留了结合RNA的能力而突变了cas13b 切割靶RNA的活性位点(即无催化活性，例如Silvana Konermann et al.,Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors,Cell,April 19,2018,Volume 173,Issue 3,P665-676或David B.T.Cox et al.,RNA editingwith CRISPR-Cas13,Science,Nov 24,2017,Vol 358,Issue 6636,P1019-1027中所述)；不失活的Cas13b会去切割RNA， Cas13b有结合并切割降解靶RNA的功能。
本申请中，所述的APOBEC 3A片段通常可以是指APOBEC 3A蛋白的全长，也可以是指APOBEC 3A的全长蛋白中的部分片段。所述的dCas13片段通常可以是指dCas13 蛋白的全长，也可以是指dCas13全长蛋白中的部分片段。dCas13Rx片段、dPspCas13b 片段等中所述的“片段”的含义亦是如此，既可指示所述蛋白的全长，也可指示所述蛋白的部分片段。
所述的dCas13Rx和dPspCas13b均为RNA结合蛋白。
本发明所提供的融合蛋白中，所述APOBEC 3A片段的氨基酸序列可以包括：a)如SEQ ID NO:85所示的氨基酸序列；或，b)与SEQ ID NO:85具有80％以上序列相似性的氨基酸序列、且具有a)所限定的氨基酸序列的功能。具体的，所述b)中的氨基酸序列具体指：如SEQ ID NO:85所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、 1-10个、1-5个、或1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO:85所示的多肽片段的功能的多肽片段，例如，可以是具有胞苷脱氨酶活性，更具体为可以是将胞嘧啶(C)编辑为尿嘧啶(U)的功能。所述b)中的氨基酸序列可与SEQ ID NO: 85具有80％、85％、90％、93％、95％、97％、或99％以上的同源性。
MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGF LHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRA FLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQ GCPFQPWDGLDEHSQALSGRLRAILQNQGN(SEQ ID NO:85)
本发明中，所述的APOBEC 3A片段通常也包括突变的APOBEC 3A片段，例如包含突变Y132D和/或H29K。
本发明所提供的融合蛋白中，所述包含突变Y132D的APOBEC3A(Y132D)片段通常指脱氨酶，所述APOBEC3A(Y132D)片段的氨基酸序列可以包括：c)如SEQ ID NO:2所示的氨基酸序列；或，d)与SEQ ID NO:2具有80％以上序列相似性的氨基酸序列、且具有c)所限定的氨基酸序列的功能。具体的，所述d)中的氨基酸序列具体指：如SEQ ID NO:2所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、1-30个、 1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、或1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO:2所示的多肽片段的功能的多肽片段，例如，可以是具有胞苷脱氨酶活性，更具体为可以是将胞嘧啶(C)编辑为尿嘧啶(U)的功能。所述d)中的氨基酸序列可与SEQ ID NO:2具有80％、85％、90％、93％、95％、97％、或99％以上的同源性。
MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGF LHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVR AFLQENTHVRLRIFAARIYDDDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDH QGCPFQPWDGLDEHSQALSGRLRAILQNQGN(SEQ ID NO:2)
本发明所提供的融合蛋白中，所述包含突变H29K的APOBEC3A(H29K)片段的氨基酸序列可以包括：e)如SEQ ID NO:90所示的氨基酸序列；或，f)与SEQ ID NO:90具有 80％、85％、90％、93％、95％、97％、或99％以上序列同源性的氨基酸序列、且具有 e)所限定的氨基酸序列的功能。
本发明所提供的融合蛋白中，所述dPspCas13b片段的氨基酸序列可以包括：g)如SEQ ID NO:1所示的氨基酸序列；或，h)与SEQ ID NO:1具有80％以上序列相似性、且具有g)所限定的氨基酸序列的功能的氨基酸序列。具体的，所述h)中的氨基酸序列具体指：如SEQID NO:1所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是 1-50、130个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10 个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO: 1所示的多肽片段的功能的多肽片段，例如，具有dPspCas13b的靶向活性，更具体可以是能够在合适的sgRNA的引导下靶向RNA的活性。所述h)中的氨基酸序列可与SEQ ID NO:1具有80％、85％、90％、93％、95％、97％、或99％以上的同源性。
MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNENNENLWFHP VMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNS NDIFEVLKRAFGVLKMYRDLTNAYKTYEEKLNDGCEFLTSTEQPLSGMINNYYTVAL RNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLH LSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSN KSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYID YGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEAETMRKQE NGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNKVEMFINDKEDSAPLLP VIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFQAMQKE EVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDD RKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIA VYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFARSIPANAVEFYERYLIER KFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELPRQMFDNEI KSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFYQWNRNYRYMDMLKGE YDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNRQMRNASSEEIETILD KRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKG ILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKED (SEQ ID NO:1)
较佳地，所述dCas13Rx片段的氨基酸序列可以包括：m)如SEQ ID NO:91所示的氨基酸序列；或，n)与SEQ ID NO:91具有80％以上序列相似性、且具有m)所限定的氨基酸序列的功能的氨基酸序列。具体的，所述n)中的氨基酸序列具体指：如SEQ ID NO: 91所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、130个、1- 20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和 /或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1- 3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO:91所示的多肽片段的功能的多肽片段，例如，具有dPspCas13b的靶向活性，更具体可以是能够在合适的sgRNA的引导下靶向RNA的活性。所述n)中的氨基酸序列可与SEQ ID NO:91具有 80％、85％、90％、93％、95％、97％、或99％以上的同源性。
较佳地，所述dPspCas13b片段位于所述APOBEC 3A片段的N端。
较佳地，所述dCas13Rx片段位于所述APOBEC 3A片段的C端或(嵌合)插入所述APOBEC 3A片段中间；例如将所述dCas13Rx片段的loop3位点(通常为所述dCas13Rx 片段的第559-586位)、所述dCas13Rx片段的第178-192位、所述dCas13Rx片段的第 377-391位替换为所述APOBEC 3A片段。通常替换时所述的APOBEC 3A片段可以是直接与所述的dCas13Rx片段相连，也可以是通过接头进行连接。在一些实施方案中，任选地在融合蛋白中的每个片段之间提供接头(Linker)。所述的接头可为本领域常规，在一些实施方案中，接头具有1-100个(或3-20个、4-15个，无限制)氨基酸残基。在一些实施方案中，接头的氨基酸残基的至少10％、20％、30％、40％、50％、60％、70％、 80％或90％是选自由丙氨酸、甘氨酸、半胱氨酸和丝氨酸组成的群组的氨基酸残基，例如如SEQ ID NO:99(GS linker：GGSGGSGGSGGSGGSGGS)所示的接头。
本申请中，所述dCas13Rx片段的loop3位点的具体位置对于本领域技术人员来说应该是已知的(DOI:https://doi.org/10.1016/j.cell.2018.09.001)，例如，可以是位于dCas13Rx 片段的558Asn～587Met之间的位置。在某一较佳实施例中，可以将559-586氨基酸片段被替换为APOBEC 3A片段，所述的APOBEC 3A片段被嵌合在dCasd之间能够提高CURE编辑的特异性，明显降低其在全转录组上的脱靶效应(这一融合蛋白被称为CURE- X)。
在某一较佳实施例中，当将所述dCas13Rx片段的loop3位点替换为所述APOBEC 3A片段时，所述dCas13Rx片段通常被分为第一dCas13Rx片段和第二dCas13Rx片段，所述第一dCas13Rx片段的氨基酸序列可以包括：i)如SEQ ID NO:3所示的氨基酸序列；或，j)与SEQID NO:3具有80％以上序列相似性、且具有i)所限定的氨基酸序列的功能的氨基酸序列。具体的，所述j)中的氨基酸序列具体指：如SEQ ID NO:3所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、130个、1-20个、1-10个、 1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C-末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQID NO:3所示的多肽片段的功能的多肽片段，例如，可以是第一dCas13Rx片段与第二dCas13Rx片段配合后依然具有dCas13Rx 的靶向活性，更具体可以是能够在合适的sgRNA的引导下靶向RNA的活性。所述j)中的氨基酸序列可与SEQ ID NO:3具有80％、85％、90％、93％、95％、97％、或99％以上的相似性。所述第一dCas13Rx片段的核苷酸序列可以是例如如SEQ ID NO:88所示。
IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSIRSVNEGEAFS AEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESA DGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVYTYDEFKDPE HHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIINYGNECYDI LALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSK NSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRK NHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVINLRGSF NDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVS AFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSA KIADELRLIKSFARMGEPIADARRAMYIDAIRILGTN(SEQ ID NO:3)
所述的第二dCas13Rx片段的氨基酸序列可以包括：k)如SEQ ID NO:4所示的氨基酸序列；或，l)与SEQ ID NO:4具有80％以上序列相似性、且具有k)所限定的氨基酸序列的功能的氨基酸序列。具体的，所述l)中的氨基酸序列具体指：如SEQ ID NO:4所示的氨基酸序列经过取代、缺失或者添加一个或多个(具体可以是1-50、130个、1-20个、 1-10个、1-5个、1-3个、1个、2个、或3个)氨基酸而得到的，或者在N-末端和/或C- 末端添加一个或多个(具体可以是1-50个、1-30个、1-20个、1-10个、1-5个、1-3个、1 个、2个、或3个)氨基酸而得到的，且具有氨基酸如SEQ ID NO:4所示的多肽片段的功能的多肽片段，例如，可以是第一dCas13Rx片段与第二dCas13Rx片段配合后依然具有dCas13Rx的靶向活性，更具体可以是能够在合适的sgRNA的引导下靶向RNA的活性。所述l)中的氨基酸序列可与SEQ ID NO:4具有80％、85％、90％、93％、95％、 97％、或99％以上的相似性。所述第二dCas13Rx片段的核苷酸序列可以是例如如SEQ ID NO:97所示。
MRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNG KNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRENAEREK FKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEKGFSSV TKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKAEEFTR QINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVHAYIND IAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKE KKKVSGNS(SEQ ID NO:4)
本发明所提供的融合蛋白中，所述的取代、缺失或者添加可以是保守氨基酸取代。所述“保守氨基酸取代”具体可以是指氨基酸残基被其他具有相似侧链的氨基酸残基取代的情况。具有相似侧链的氨基酸残基家族对于本领域技术人员来说应该是已知的，例如，可以是包括但不限于碱性侧链(例如赖氨酸，精氨酸，组氨酸)，酸性侧链(例如天冬氨酸，谷氨酸)，不带电荷的极性侧链(例如，甘氨酸，天冬酰胺，谷氨酰胺，丝氨酸，苏氨酸，酪氨酸，半胱氨酸)，非极性侧链(例如丙氨酸，缬氨酸，亮氨酸，异亮氨酸，脯氨酸，苯丙氨酸，甲硫氨酸，色氨酸)异亮氨酸)和芳族侧链(例如酪氨酸，苯丙氨酸，色氨酸，组氨酸)等家族。保守型氨基酸取代更具体可以包括但不限于下表中所列的具体情况，表1(氨基酸相似度矩阵)中的数字表示两个氨基酸之间的相似度，当数字大于等于0时认为是保守氨基酸取代，表2为示例性的保守氨基酸取代的方案。
表1
C G P S A T D E N Q H K R V M I L F Y W
W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6
I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6
K -5 -2 -1 0 -1 0 0 0 1 1 0 5
H -3 -2 0 -1 -1 -1 1 1 2 3 6
Q -5 -1 0 -1 0 -1 2 2 1 4
N -4 0 -1 1 0 0 2 1 2
E -5 0 -1 0 0 0 3 4
D -5 1 -1 0 0 0 4
T -2 0 0 1 1 3
A -2 1 1 1 2
S 0 1 1 1
P -3 -1 6
G -3 5
C 12
表2
Figure BDA0002611487570000081
Figure BDA0002611487570000091
较佳地，所述的融合蛋白还包括一个或多个NLS(核定位信号或序列)或其突变，例如包括一个或两个NLS或其突变。所述NLS的主要功能是入核。所述的突变是指失去核定位功能的NLS。通常，该信号由暴露于蛋白质表面的一个或多个带正电荷的赖氨酸或精氨酸的短序列组成。不同的核定位蛋白可能共享相同的NLS。NLS具有与核输出信号(NES)相反的功能，后者将蛋白质靶向于核外。其中，所述NLS例如可以为SV40 NLS(猿猴病毒40Simianvirus 40核定位信号)和/或bpNLS(Bipartite NLS)。
其中，所述NLS优选可以位于所述dCas13片段的N端或C端，或可以位于所述APOBEC 3A片段的N端或C端，或可以位于所述dCas13片段与所述APOBEC 3A片段之间。在某一较佳实施例中，优选至少一个NLS或其突变位于所述融合蛋白的N端。在某一较佳实施例中，至少一个NLS或其突变位于所述融合蛋白的C端。在某一较佳实施例中，至少一个NLS或其突变(例如两个NLS或其突变)位于所述融合蛋白的中间。在某一较佳实施例中，至少一个NLS或其突变分别位于所述融合蛋白的N端和C端。在某一较佳实施例中，至少一个NLS或其突变分别位于所述融合蛋白的N端和C端以及插入所述融合蛋白的中间。在某一较佳实施例中，所述NLS片段可以位于dPspCas13b的 N端，也可以位于APOBEC3A的C端；同样可以在CURE-X里位于第一dCas13Rx片段的N端，也可以位于所述第一dCas13Rx片段与APOBEC3A(Y132D)片段之间，也可以位于APOBEC3A(Y132D)片段与第二dCas13Rx片段之间，也可以位于第二dCas13Rx片段的C端。
所述NLS片段可以包括如SEQ ID NO:5所示的氨基酸序列。
KRTADGSEFESPKKKRKV(SEQ ID NO:5)
本发明所提供的融合蛋白中，所述融合蛋白还可以包括mNLS(突变的核定位序列，相当于一段肽段，不行使进核功能)片段。所述NLS片段可以位于dPspCas13b的N端，所述mNLS片段可以包括如SEQ ID NO:6所示的氨基酸序列。
KRTADGSEFESPKAAAK(SEQ ID NO:6)
较佳地，所述的融合蛋白还包括NES(核输出信号)片段，例如HIV NES片段。
较佳地，所述的融合蛋白用于碱基编辑，优选用于C到U的碱基编辑。
在某一较佳实施例中，所述融合蛋白自N端至C端依次包括NLS或其突变、dPspCas13b片段和APOBEC 3A片段(可以是包含突变Y132D和/或H29K的APOBEC 3A片段)。在某一较佳实施例中，所述融合蛋白自N端至C端依次由NLS或其突变、 dPspCas13b片段和APOBEC 3A片段(可以是包含突变Y132D和/或H29K的APOBEC 3A片段)组成。
在某一较佳实施例中，所述融合蛋白自N端至C端依次包括NLS(或其突变)、dPspCas13b片段、APOBEC 3A片段(可以是包含突变Y132D和/或H29K的APOBEC 3A片段)、和NLS(或其突变)。在某一较佳实施例中，所述融合蛋白自N端至C端依次由NLS(或其突变)、dPspCas13b片段、APOBEC 3A片段(可以是包含突变Y132D 和/或H29K的APOBEC 3A片段)、和NLS(或其突变)组成。
在某一较佳实施例中，所述融合蛋白自N端至C端依次包括NLS或其突变、dPspCas13b片段、HIV NES片段和APOBEC 3A片段(可以是包含突变Y132D和/或 H29K的APOBEC 3A片段)。在某一较佳实施例中，所述融合蛋白自N端至C端依次由NLS、dPspCas13b片段、HIV NES片段和APOBEC 3A片段(包含突变Y132D)组成。在某一较佳实施例中，所述融合蛋白自N端至C端依次由突变的NLS、dPspCas13b 片段、HIV NES片段和APOBEC 3A片段(包含突变Y132D)组成。
在某一较佳实施例中，所述融合蛋白自N端至C端依次包括NLS(或其突变)、dPspCas13b片段、HIV NES片段、APOBEC 3A片段(可以是包含突变Y132D和/或H29K 的APOBEC 3A片段)、和NLS(或其突变)。在某一较佳实施例中，所述融合蛋白自 N端至C端依次由NLS(或其突变)、dPspCas13b片段、HIV NES片段、APOBEC 3A 片段(可以是包含突变Y132D和/或H29K的APOBEC 3A片段)、和NLS(或其突变) 组成。
在某一较佳实施例中，当所述dCas13Rx片段插入所述APOBEC 3A片段中间时，所述dCas13Rx片段被分为第一dCas13Rx片段和第二dCas13Rx片段，所述融合蛋白自N 端至C端依次包括第一dCas13Rx片段、APOBEC 3A片段和第二dCas13Rx片段。在某一较佳实施例中，当在所述dCas13Rx片段的loop3位点插入所述APOBEC 3A片段时，所述融合蛋白自N端至C端依次由第一dCas13Rx片段、APOBEC 3A片段和第二 dCas13Rx片段组成。
在某一较佳实施例中，所述融合蛋白自N端至C端依次包括NLS或其突变、第一dCas13Rx片段、NLS或其突变、APOBEC 3A片段(可以是包含突变Y132D和/或H29K 的APOBEC3A片段)、NLS或其突变、第二dCas13Rx片段、和NLS或其突变。在某一较佳实施例中，所述融合蛋白自N端至C端依次由NLS或其突变、第一dCas13Rx片段、NLS或其突变、APOBEC 3A片段(可以是包含突变Y132D和/或H29K的APOBEC 3A片段)、NLS或其突变、第二dCas13Rx片段、和NLS或其突变组成。
在某一较佳实施例中，当所述dCas13Rx片段插入所述APOBEC 3A片段中间时，所述dCas13Rx片段被分为第一dCas13Rx片段和第二dCas13Rx片段，所述融合蛋白自N 端至C端依次包括NLS、第一dCas13Rx片段、NLS、接头、APOBEC 3A片段、接头、 NLS、第二dCas13Rx片段、和NLS。
在某一较佳实施例中，所述融合蛋白自N端至C端依次包括NLS、APOBEC 3A片段、接头、dCas13Rx片段和NLS。
本发明所提供的某一实施方案中，融合蛋白CURE-C1自N端至C端依次包括bpNLS片段、dPspCas13b片段、HIV NES片段和APOBEC 3A(Y132D)片段；CURE-C2自N端至C端依次包括突变的bpNLS片段、dPspCas13b片段、HIV NES片段和APOBEC 3A(Y132D)片段；CURE-N自N端至C端依次包括bpNLS片段、dPspCas13b片段、HIV NES片段和APOBEC 3A(Y132D)片段以及bpNLS片段；CURE-X自N端至C端依次包括bpNLS片段、氨基酸序列如SEQ ID NO:3所示的第一dCas13Rx片段、bpNLS片段、 APOBEC3A(Y132D)片段、bpNLS片段、氨基酸序列如SEQ ID NO:4所示的第二dCas13Rx 片段和bpNLS片段。
CURE-X1自N端至C端依次包括bpNLS片段、dCas13Rx片段的第1-177位、bpNLS 片段、接头、APOBEC3A(Y132D)片段、接头、bpNLS片段、dCas13Rx片段的第193-967 位和bpNLS片段。
CURE-X2自N端至C端依次包括bpNLS片段、dCas13Rx片段的第1-376位、bpNLS 片段、接头、APOBEC3A(Y132D)片段、接头、bpNLS片段、dCas13Rx片段的第392-967 位和bpNLS片段。
CURE-X3自N端至C端依次包括bpNLS片段、氨基酸序列如SEQ ID NO:3所示的第一dCas13Rx片段、bpNLS片段、接头、APOBEC3A(Y132D)片段、接头、bpNLS片段、氨基酸序列如SEQ ID NO:4所示的第二dCas13Rx片段和bpNLS片段。
CURE-X4自N端至C端依次包括bpNLS片段、APOBEC3A(Y132D)片段、接头、 dCas13Rx片段和bpNLS片段。
较佳地，所述融合蛋白的氨基酸序列包括如SEQ ID NO:7-10、93-96任一项所示的氨基酸序列；编码所述融合蛋白的核苷酸序列优选如SEQ ID NO:17、20、25、28、31、 34、48任一项所示。
在某一具体实施例中，所述融合蛋白的氨基酸序列包括CURE-C1如SEQ ID NO:7所示的氨基酸序列，CURE-C2如SEQ ID NO:8所示的氨基酸序列，CURE-X如SEQ ID NO:9所示的氨基酸序列，CURE-N如SEQ ID NO:10如所示的氨基酸序列。CURE-X如 SEQ ID NO:93所示的氨基酸序列，CURE-X如SEQ ID NO:94所示的氨基酸序列，CURE- X如SEQ ID NO:95所示的氨基酸序列，CURE-X如SEQ ID NO:96所示的氨基酸序列。
KRTADGSEFESPKKKRKVNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVA DIEGEQNENNENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQR EYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNAYKTYEEKLNDGCEFLTST EQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFF LSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFG INSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLM KRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLN GFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNK VEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVD VHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVD DMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDG ENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFAR SIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYS EDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFYQ WNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSN RQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDG ERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIG NLLELVGSDIVSKEDGSLQLPPLERLTLGSGSSMEASPASGPRHLMDPHIFTSNFNNGIG RHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQ LDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDDDPLYKEALQ MLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQ GN(SEQ ID NO:7)
KRTADGSEFESPKAAAKVNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVA DIEGEQNENNENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQR EYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNAYKTYEEKLNDGCEFLTST EQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFF LSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFG INSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLM KRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLN GFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNK VEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVD VHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVD DMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDG ENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFAR SIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYS EDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFYQ WNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSN RQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDG ERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIG NLLELVGSDIVSKEDGSLQLPPLERLTLGSGSSMEASPASGPRHLMDPHIFTSNFNNGIG RHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQ LDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDDDPLYKEALQ MLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQ GN(SEQ ID NO:8)
MKRTADGSEFESPKKKRKVGSIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARL EKIVEGDSIRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQ DMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDI IGFGKFSTVYTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAF FSKEGRNYIINYGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITK LREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDN EKSLSEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKK KDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLI GVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNGTSKR TADGSEFESPKKKRKVGPGSMEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVE RLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWF ISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDDDPLYKEALQMLRDAGAQVSIM TYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGNGSKRTADGSEFESPKKKRKVGSMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTG RENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKL EEKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDE KKAEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVAR YVHAYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLK LLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNSGSKRTADGSEFESPKKKR KV(SEQ ID NO:9)
KRTADGSEFESPKKKRKVNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVA DIEGEQNENNENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQR EYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNAYKTYEEKLNDGCEFLTST EQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFF LSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFG INSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLM KRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLN GFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNK VEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVD VHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVD DMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDG ENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFAR SIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYS EDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFYQ WNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSN RQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDG ERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIG NLLELVGSDIVSKEDGSLQLPPLERLTLGSGSSMEASPASGPRHLMDPHIFTSNFNNGIG RHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQ LDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDDDPLYKEALQ MLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQ GNAAGSKRTADGSEFESPKKKRKV(SEQ ID NO:10)
为了解决上述技术问题，本发明第二方面提供一种分离的多核苷酸，其编码本发明第一方面所提供的融合蛋白。
较佳地，其核苷酸序列如SEQ ID NO:17、20、25、28、31、34、48任一项所示。
为了解决上述技术问题，本发明第三方面提供了一种构建体，所述构建体含有本发明第二方面所提供的分离的多核苷酸。所述构建体通常可以通过将所述分离的多核苷酸插入合适的表达载体中构建获得，本领域技术人员可选择合适的表达载体，例如，所述表达载体可以是包括但不限于细菌表达载体、真菌表达载体、动物表达载体(例如，昆虫、果蝇、线虫、鱼类、斑马鱼、哺乳动物、小鼠、大鼠、兔、猪、猴、人等)、植物表达载体等。
为了解决上述技术问题，本发明第四方面提供一种表达系统，所述表达系统含有本发明第三方面所提供的构建体或基因组中整合有(可以是外源的)本发明第二方面所提供的分离的多核苷酸。所述表达系统可以是宿主细胞，所述宿主细胞可以表达如上所述的融合蛋白，所述融合蛋白可以与sgRNA相配合，从而可以将所述融合蛋白定位到目标区域，实现目标区域的碱基编辑。在本发明另一具体实施例中，所述宿主细胞可以是真核细胞或原核细胞，更具体可以是人细胞，更具体可以是人肾上皮细胞系，更具体可以是293T。
为了解决上述技术问题，本发明第五方面提供了一种融合蛋白的制备方法，其包括以下步骤：
(1)获得本发明第四方面所述的表达系统；
(2)筛选所述表达系统，表达并纯化所述融合蛋白。
为了解决上述技术问题，本发明第六方面提供了一种碱基编辑器(又可称为碱基编辑系统)，其包括本发明第一方面所提供的融合蛋白、本发明第二方面所述的多核苷酸、本发明第三方面所述的构建体和/或本发明第四方面所述的表达系统。所述碱基编辑体系还可以包括指导RNA(sgRNA)。本领域技术人员可以根据基因的目标编辑区域，选择合适的靶向目标区域的sgRNA。
其中，所述指导RNA可以优选为包括28-36个碱基，优先为32个碱基。
其中，所述指导RNA可以优选其第5位不为C。
其中，所述指导RNA的序列可以优选如SEQ ID NO:92所示。
在某一较佳实施例中，本申请的融合蛋白(例如CURE-C1、CURE-C2、CURE-N) 可用于编辑核酸序列的编辑框里碱基范围的选择比较广内的可以选择6-20个碱基，例如为8、10、12、14或18个碱基，也可以选择为多于20个碱基。在某一较佳实施例中，本发明的融合蛋白(例如CURE-X)的编辑框优选选择10-14个碱基数。
在某一较佳实施例中，用于编辑的核酸序列的编辑框为14个碱基组成的环，所述指导RNA优选为包括28-36个碱基，优先为32个碱基时效果更佳。
本申请中，所述的编辑框通常是指待编辑的核酸序列中主要需要编辑的区域，该区域通常能在引导RNA的作用下在靶位点诱导形成一个环状结构。
本申请中，所述sgRNA的序列通常可以与目标区域至少部分互补，从而可以与所述融合蛋白相配合，将所述融合蛋白定位到目标区域，实现目标区域的碱基编辑，具体可以是腺嘌呤脱氨基反应，即将胞嘧啶(C)编辑为尿嘧啶(U)。本发明所提供的碱基编辑体系可以由sgRNA与靶基因互补，使得靶基因在编辑的位置产生环状结构下实现编辑，并在sgRNA使得靶基因在编辑位置产生不同碱基数量的环状结构使得编码框在一定条件下有现存C>URNA碱基编辑所特有的编辑。例如，在32nt(碱基)sgRNA的核苷酸序列的长度情况下，与靶基因互补配对，使得靶基因编辑位置产生不同长度的环状结构都有很好的编辑(例如，6-20nt)均有良好的编辑活性，再例如，在sgRNA的核苷酸序列的长度为32nt的情况下，靶基因编辑位置形成6-20nt的环状结构，CURE-C1的编辑效率高达约40％-60％；在sgRNA的核苷酸序列的长度为28nt的情况下，靶基因编辑位置形成8-20nt的环状结构，CURE-X的编辑效率约15％-50％，其中8、12、14nt时高达 40％-50％，10、18、20nt时的编辑效率约为20％-30％。
较佳地，所述碱基编辑器用于真核生物的碱基编辑。
较佳地，所述碱基编辑器用于体外的碱基编辑。
较佳地，所述碱基编辑器用于编辑核酸序列上的胞嘧啶(C)，通常是将C编辑为U(尿嘧啶)。
较佳地，所述碱基编辑器中使用的荧光报告系统包括第一质粒和第二质粒，所述第一质粒的序列如SEQ ID NO:89所示，所述第二质粒的序列如SEQ ID NO:101所示；其组成结构详见图2。
本发明第七方面还提供了一种试剂盒，其包括本发明第一方面所提供的融合蛋白、本发明第二方面所述的多核苷酸、本发明第三方面所述的构建体、本发明第四方面所述的表达系统和/或本发明第六方面所述的碱基编辑器。
较佳地，所述试剂盒还包括指导RNA；所述指导RNA优选为包括28-36个碱基，优先为32个碱基；所述指导RNA优选其第5位不为C；所述指导RNA的序列更优选如 SEQ ID NO:92所示。
较佳地，所述试剂盒用于编辑核酸序列的编辑框，所述编辑框优选包括6-20个碱基，例如为8、10、12、14或18个碱基。
较佳地，所述试剂盒用于真核生物的碱基编辑。
较佳地，所述试剂盒用于体外的碱基编辑。
较佳地，所述试剂盒用于编辑核酸序列上的胞嘧啶(C)，通常是将C编辑为U。
较佳地，所述碱基编辑器中使用的荧光报告系统包括第一质粒和第二质粒，所述第一质粒的序列如SEQ ID NO:89所示，所述第二质粒的序列如SEQ ID NO:101所示；其组成结构详见图2。
本发明第八方面提供本发明第一方面所提供的融合蛋白、或本发明第二方面所提供的分离的多核苷酸、或本发明第三方面所提供的构建体、或本发明第四方面所提供的表达系统、或本发明第六方面所提供的碱基编辑器、或本发明第七方面所提供的试剂盒在碱基编辑中的用途，优选为真核生物的基因表达中的用途，所述真核生物具体可以是后生动物，具体可以是包括但不限于人细胞等。所述用途具体可以是包括但不限于修复由 C到U的基因突变、恢复由单碱基突变产生错义突变或者没能正确终止的失活蛋白的翻译，改变RNA剪辑等。在本发明一具体实施例中，被编辑的基因可以是PGAP2(Gene ID：27315)。在本发明一具体实施例中，所述用途为在体外的碱基编辑中的用途。
本发明第九方面提供一种碱基编辑方法，包括：通过本发明第一方面所提供的融合蛋白、或本发明第二方面所提供的分离的多核苷酸、或本发明第三方面所提供的构建体、或本发明第四方面所提供的表达系统、或本发明第六方面所提供的碱基编辑器、或本发明第七方面所提供的试剂盒进行碱基编辑。例如，所述碱基编辑方法可以包括：在适当条件下培养本发明第四方面所提供的表达系统，从而表达所述融合蛋白，所述融合蛋白可以在与其配合的靶向目标区域的sgRNA存在的条件下，对靶标区域进行碱基编辑。提供所述sgRNA存在的条件的方法对于本领域技术人员来说应该是已知的，例如，可以是在适当条件下培养能够表达所述sgRNA的表达系统，所述表达系统可以是包括含有编码所述sgRNA的多核苷酸的表达载体的宿主细胞、或染色体中整合有编码所述sgRNA的多核苷酸的宿主细胞。在本发明一具体实施例中，所述碱基编辑为体外碱基编辑。在本发明一具体实施例中，所述碱基编辑为真核生物的碱基编辑。在本发明一具体实施例中，所述碱基编辑为将C编辑为U的碱基编辑。在本发明一具体实施例中，所述方法包括使待碱基编辑的样品与指导RNA(例如本发明第七方面所述的指导RNA)与所述的融合蛋白、所述的多核苷酸、所述的构建体、所述的表达系统、所述的碱基编辑器、或所述的试剂盒接触以进行碱基编辑。
本发明另一方面还提供了一种荧光报告系统(具体结构见图2)，其包括第一质粒和第二质粒，所述第一质粒的序列如SEQ ID NO:89所示，所述第二质粒的序列如SEQ IDNO:101所示。
本发明另一方面还提供了上述的荧光报告系统在检测上述的融合蛋白的编辑效率中的应用。
为了简便，本文仅明确地公开了一些数值范围。然而，任意下限可以与任何上限组合形成未明确记载的范围；以及任意下限可以与其它下限组合形成未明确记载的范围，同样任意上限可以与任意其它上限组合形成未明确记载的范围。此外，尽管未明确记载，但是范围端点间的每个点或单个数值都包含在该范围内。因而，每个点或单个数值可以作为自身的下限或上限与任意其它点或单个数值组合或与其它下限或上限组合形成未明确记载的范围。
在本文的描述中，需要说明的是，除非另有说明，“以上”、“以下”为包含本数，“一种或多种”中“多种”的含义是两种以上。
本发明中，所述“包括或包含”可以是指除了包括后面所列举的成分，还存在其他成分；也可以是指“由……组成”，即只包括后面所列举的成分而不存在其他成分。
本发明的上述发明内容并不旨在描述本发明中的每个公开的实施方式或每种实现方式。如下描述更具体地举例说明示例性实施方式。在整篇申请中的多处，通过一系列实施例提供了指导，这些实施例可以以各种组合形式使用。在各个实例中，列举仅作为代表性组，不应解释为穷举。
本发明中，所述的碱基A、C、U、I等等具有本领域技术人员通常所理解的含义，例如A通常是指腺嘌呤，C通常是指胞嘧啶，U通常是指尿嘧啶，I通常是指次黄嘌呤。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明所提供的融合蛋白，所基于的CRISPR系统中dPspCas13b和dCas13Rx能在引导RNA的作用下靶向RNA，将靶位点局部环化进行C 到U的编辑，降低了RNA碱基编辑脱靶的同时具有APOBEC3A的胞嘧啶脱氨作用。本发明的融合蛋白用于碱基编辑时并不会破坏任何腺嘌呤，没有A到I的脱靶，并且该系统更倾向于编辑UC，也能编辑UCC里的CC，最终能变为UUU，对内源性转录本的编辑中UC序列的编辑效率较高。此外，特异性也显著提高。综上所述，本发明利用CRISPR 系统中能够特异靶向RNA的C>U的编辑编辑，不仅能给与C>U碱基编辑提供了新的选择，而且也跟目前仅有的RNA的C>U的编辑编辑器RESCUE-S互补，在一定条件下，扩宽了C>U RNA碱基编辑的试用范围，如能同时编辑UCC中的后两个CC，使脯氨酸 (pro)能直接突变成苯丙氨酸(phe)或是亮氨酸(lue)，也能同时或者选择性的编辑有 crRNA是的靶位点形成特殊环状发夹结构中的多个UC进行编辑。其中crRNA设计的巧妙性，也给CRISPR系统的sgRNA针对靶基因的设计提供新思路。
在本发明某一较佳实施例中，碱基编辑器CURE-C2在PPIB(I18I)、TYMS(I262I)、ACTB(I75I)基因上的编辑效率高达40％-60％，而现有的RESCUE-S在这几个基因上的编辑效率为10％-40％。
在本发明某一较佳实施例中，所使用的dCas13Rx只有954个氨基酸，是目前CRISPR系统类型VI-A，VI-B，VI-C中最小的一种，对后续通过包装AAV病毒，实现成体治疗有极大价值。另外，dCas13Rx蛋白能够在删减一些氨基酸片段后，仍然保留活性，因此将APOBEC3A(Y132D)插入dCas13Rx蛋白中间，嵌合表达的dCas13Rx(1-558)- APOBEC3A(Y132D)-dCas13Rx(587-966)蛋白仍然具有dCas13Rx的靶向RNA活性，以及 APOBEC3A(Y132D)突变靶位点的脱氨作用。通过借助CRISPR系统中能够靶向RNA的 Cas蛋白——dCas13Rx，将APOBEC3A(Y132D)融合在dCas13Rx蛋白中间位置，融合蛋白再在sgRNA的引导下，利用dCas13Rx对RNA的精确靶向功能实现目的位点定位，被包裹在dCas13Rx中间的ADAR再进行碱基C到U的脱氨突变。
在本发明某一较佳实施例中，包裹在APOBEC3A(Y132D)蛋白两端的dCas13Rx能够阻碍APOBEC3A(Y132D)对非靶向RNA的结合，从而降低非靶向性的脱靶效应，借助 CRISPR系统达到既靶向RNA又能降低RNA碱基编辑脱靶的目的。
在本发明某一较佳实施例中，所述的融合蛋白所基于的CRISPR系统中PspCas13B能够在RNA引导下靶向RNA。CURE-C与CURE-N是目前碱基编辑领域C>U的单碱基编辑中有些位点能编辑且编辑最好的editor，例如，CURE-C2在 PPIB(I18I),TYMS(I262I),ACTB(I75I)基因上的编辑效率高达40％-60％而RESCUE-S在这几个基因上的编辑效率为10％-40％。CURE-C与CURE-N不仅没有A到I的脱靶，而且还能使UCC为编辑UUU,直接能将Pro(脯氨酸)变为Phe(苯丙氨酸)或Lue(亮氨酸)。因此将APOBEC3A(Y132D)插入PspCas13b蛋C端，融合表达的PspCas13B- APOBEC3A(Y132D)蛋白仍然具有PspCas13b的靶向RNA活性，以及APOBEC3A(Y132D) 突变靶位点的脱氨作用。通过借助CRISPR系统中能够靶向RNA的Cas蛋白——PspCas13b，将APOBEC3A(Y132D)融合在dPspCas13b蛋白C端，融合蛋白再在sgRNA 的引导下，利用dPspCas13b对RNA的精确靶向功能实现目的位点定位，利用APOBEC3A 识别RNA的特性(识别环状RNA)，巧妙利用sgRNA与靶碱基编辑位点6-20nt以外的序列相互补，以此使靶基因上编辑位点暴露形成APOBEC3A所识别6-20nt的环状结构，以此来对编辑位点里的TC选择性的编辑，例如CURE-C1可选择性的编辑PGAP2基因里的相邻两个TC中的一个或者两个TC。借助CRISPR系统达到既靶向RNA又能对暴露出来的编辑框进行TC选择性编辑。
附图说明
图1为示意图，其中上图为CURE载体示意图，下图为APOBEC 3A经典的编辑结构。
图2为报告基因荧光报告系统设计图。
图3为指导RNA(sgRNA)诱导荧光报告基因转录子形成的环状结构，其中编辑的位点为UAUC中的C。
图4为CURE-C1在荧光报告基因上编辑效率的结果，图A和B为Facs结果，图C 为sanger结果。
图5为CURE-C1在在荧光报告基因上编辑效率的NGS结果，其中图A为on-target 的sgRNA与其他sgRNA的结构图，图B为各个sgRNA编辑效率的deep-seq分析测序结果。
图6为CURE-C1编辑内源碱基编辑效率sanger测序结果与deep-seq测序分析结果比较。
图7为CURE-C1和CURE-X在不同长度sgRNA引导下在报告系统上的编辑效率的测序结果。左图为sgRNA在报告基因上的位置，其中UAUC在状结构图的正中间；右图为编辑效率的测序结果，其中CURE-C1是sanger测序结果，CURE-X是deep-seq测序分析结果。
图8为CURE-C1在长度为32nt的sgRNA引导下和CURE-X在长度为28nt的sgRNA 引导下报告系统形成不同长度的环状结构的编辑效率的测序结果。左图为不同长度的环状结构图，其中UAUC在状结构图的正中间；右图为编辑效率的测序结果，其中CURE- C1是sanger测序结果，CURE-X是deep-seq测序分析结果。
图9为CURE-C1在长度为32nt的sgRNA引导下和CURE-X在长度为28nt的sgRNA 引导下报告系统形成14nt环状结构的下编辑UC在环状结构里的不同位置的编辑效率的测序结果。左图UAUC编辑位点的位置，右图为编辑效率的测序结果，其中CURE-C1是 sanger测序结果，CURE-X是deep-seq测序分析结果。
图10为CURE-C1在长度为32nt的sgRNA引导下和CURE-X在长度为28nt的 sgRNA引导报告系统形成14nt环状结构的下的测序结果。左图UC编辑位点两边不同碱基的位置，右图为编辑效率的测序结果，其中CURE-C1是sanger测序结果，CURE-X是 deep-seq测序分析结果。
图11a为不同sgRNA引导下外源基因形成不同环状结构的结构图，其中同个环里可能包含两个编辑UC位点,这里暂把右边的UC定为实际编辑的位点，而左边的UC为额外编辑的位点。
图11b为CURE-C1在图11a上的编辑效率结果，左图为sanger分析结果图，右图为左图其中几个值的sanger测序结果ab1图。
图11c为CURE在图11a上的deep-seq测序分析结果，上图为CURE-C1分析结果图，右图CURE-X分析结果图。
图12为CURE与RESCUE-S在外源基因上靶向UCC的编辑效率，其中右图为CURE 和RESCUE-S的sgRNA分别在UCC外源基因上的位置；左图为deep-seq测序分析结果。
图13为CURE与RESCUE-S在内源转录本上靶向UC的编辑效率的deep-seq测序分析结果比较，其中高亮的基因为RESCUE-S文章里的位点。
图14为CURE与RESCUE-S-N在内源靶向细胞核内转录本UC的编辑效率的deep- seq测序分析结果比较，其中RESCUE-S-N是RESCUE-S核定位版本。
图15为CURE和RESCUE-S在荧光报告系统中在sgRNA引导时的全转录组脱靶情况，其中CURE-C2和CURE-N的sgRNA是32-14，CURE-X的sgRNA是28-14，RESCUE- S的sgRNA是30/22。
图16为使用了有其他的胞苷脱氨酶APOBEC 3G、APOBEC A1、APOBEC mA1、 APOBECA2的实验结果图。
图17为使用了有其他的胞苷脱氨酶APOBEC 3G、APOBEC A1、APOBEC mA1、APOBECA2的实验结果图。
图18为包含不同突变体的APOBEC 3A片段的实验结果图。
图19a为Abudayyeh et al.,2019中的结果图。
图19为根据Abudayyeh et al.,2019里的原图分析之后算出来的实验数据整合成的表格。
图20为根据Abudayyeh et al.,2019里的原图分析之后算出来的实验数据整合成的表格。
图21为CURE RNA碱基编辑器在基因组上R-环(loop)的脱靶情况。
图22为使用其他RNA结合蛋白MCP和CIRTS的实验结果图，上图是MCP-A3A 的sanger ab1图，下图是CIRTS-A3A的sanger结果。
图23显示了将dCas13Rx置于APOBEC 3A的C端或其他位置时的结果图。
具体实施方式
为了使本发明的发明目的、技术方案和有益技术效果更加清晰，以下结合实施例对本发明进行进一步详细说明，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。应当理解的是，本说明书中描述的实施例仅仅是为了解释本发明，并非为了限定本发明。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
应当理解地是，下列实施例中未具体注明的工艺设备或装置均采用本领域内的常规设备或装置。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA 技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook 等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring HarborLaboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS INMOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；theseries METHODS IN ENZYMOLOGY，Academic Press，San Diego； Wolffe，CHROMATINSTRUCTURE AND FUNCTION，Third edition，Academic Press， San Diego，1998；METHODSIN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，AcademicPress，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，ChromatinProtocols(P.B.Becker，ed.)Humana Press，Totowa，1999 等。
实施例1
CURE系统质粒的构建：
pC0053-CMV-dPspCas13b-GS-ADAR2DD(E488Q)-delta-984-109购于Addgene(plasmid#103869；http://n2t.net/addgene:103869；RRID:Addgene_103869)。将 4276F(GGCGTAATCATGGTCATAGCTG)(SEQ ID NO:11)和 4276R(GCTCGAGCCGGATCCCAGTGTCAGTCTTTCAA)(SEQ ID NO:12)引物对稀释到10μM，再从pC0053模板上使用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)扩增 dPspCas13b以及细菌E.coli载体片段。将4191F(GGATCCGGCTCGAGCATGGAAGCCAGCCCAGCA)(SEQ ID NO:13)和 4191R(ATCCaGCGGCCGCGTTTCCCTGATTCTGGAGAATG)(SEQ ID NO:14)引物对稀释到10μM，再从人的基因组上使用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)扩增 Apobec 3A片段。
将4275F(ACGCGGCCGCTGGATCCGCTACTAACTTCAGCC)(SEQ ID NO:15)和 4275R(GACCATGATTACGCCTtCGAGGTCGACGGTATCGATG)(SEQ ID NO:16)引物对稀释到10μM，再从TC243上使用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)扩增 mcherry和WPRE片段(SEQ ID NO:86)。PCR扩增产物经通过AxyPrep PCR Clean-up 试剂盒(Axygen,AP-PCR-500G)纯化回收后，上述三个片段Apobec 3A片段、mcherry 和WPRE片段用
Figure BDA0002611487570000232
HiFi DNA AssemblyMaster Mix(E2621L)重组。重组产物在 50℃孵育15min后转化涂板，经Sanger测序得到正确的没有polyA的质粒TC979：CMV dPspCas13b-Apobec3A序列(SEQ ID NO:17)。
以TC979：CMV dPspCas13b-Apobec3A质粒作为载体，用BspQI(NEB，R0712S) 和EcoRV-HF(NEB，R3195S)将mcherry后面的WPRE切下来，酶切的载体(7276bp) 用MonarchDNA Gel Extraction Kit(NEB，T1020L)试剂盒回收。将4676F (gcatgcaagcttgatAATCAACCTCTGGATTACAAAATTTG)(SEQ ID NO:18)和 4676R(agtgagcgaggaagcTCCCCAGCATGCCTG)(SEQ ID NO:19)引物对稀释到10μM，从TC332上使用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)扩增WPRE和PolyA片段 (SEQ ID NO:87)。PCR扩增产物经通过AxyPrep PCR Clean-up试剂盒(Axygen，AP- PCR-500G)纯化回收后，上述两个片段用
Figure BDA0002611487570000231
HiFi DNAAssembly Master Mix(E2621L)重组。重组产物在50℃孵育15min后转化涂板，经Sanger测序得到正确的有polyA的质粒TC1116：CMV dPspCas13b-Apobec3A序列(SEQ ID NO:20)
以TC1116：CMV dPspCas13b-Apobec3A质粒作为载体，用NotI-HF(NEB，R3189S) 和XhoI(NEB，R0146V)将Apobec3A切下来，酶切的载体(7488bp)用Monarch DNA GelExtraction Kit(NEB,T1020L)试剂盒回收。将4531F(CACTGGGATCCGGCTCGA)(SEQ ID NO:21)和4543R(ATCATAGATGCGGGCAG)(SEQ ID NO:22)引物对稀释到10μM，从TC1116上使用诺唯赞高保真酶试剂盒(Vazyme，p501-d2)扩增一半Apobec3A片段。
将4539F(TGCCCGCATCTATGATGACGACCCCCTA)(SEQ ID NO:23)和 4544R(AGCAGGCTGAAGTTAGTAG)(SEQ ID NO:24)引物对稀释到10μM，从TC1116 上使用诺唯赞高保真酶试剂盒(Vazyme，p501-d2)扩增另外一半引入Y132D突变的 Apobec3A片段，两段PCR扩增产物经通过AxyPrep PCR Clean-up试剂盒(Axygen,AP- PCR-500G)纯化回收后，上述三个片段用
Figure BDA0002611487570000241
HiFi DNA Assembly Master Mix(E2621L)重组。重组产物在50℃孵育15min后转化涂板，经Sanger测序得到正确的的质粒TC1132N：CMV dPspCas13b-Apobec3A(Y132D)序列(SEQ ID NO:25)。
将5518F(TTCGAATCCCCTAAAAAGAAAAGAAAGGTGaacatccccgctctggtgg)(SEQ ID NO:26)和5518R(CTCGGATCCATCGGCGGTGCGCTTcatggtggcaagcttaagtttaaacgcta) (SEQ ID NO:27)引物对稀释到10μM，从TC1132N上使用诺唯赞高保真酶试剂盒 (Vazyme,p501-d2)环p质粒，引物上带上bpNLS。PCR扩增产物经通过AxyPrep PCR Clean-up试剂盒(Axygen,AP-PCR-500G)纯化回收后，用KLD Enzyme Mix(NEB,M0554S) 连接。室温孵育5min后转化涂板，经Sanger测序得到editor的C端添加有bpNLS质粒 (CURE-C1)，TC1309：CMV bpNLS-dPspCas13b-Apobec3A(Y132D)序列(SEQ ID NO: 28)。
将6728F(GTGaacatccccgctctggtgg)(SEQ ID NO:29)和 6728R(CTTTgcagcagcTTTAGGGGATTCGAACTCGGA)(SEQ ID NO:30)引物对稀释到 10μM，从TC1309上使用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)环p质粒，引物上带上突变的bpNLS。PCR扩增产物经通过AxyPrep PCR Clean-up试剂盒(Axygen，AP- PCR-500G)纯化回收后，用KLD Enzyme Mix(NEB,M0554S)连接。室温孵育5min后转化涂板，经Sanger测序得到editor的C端有突变的bpNLS质粒(CURE-C2)，TC1681： CMV-bpNLS(mutant)-dPspCas13b-Apobec3A(Y132D)序列(SEQ ID NO:31)。
以TC1309：CMV bpNLS-dPspCas13b-Apobec3A(Y132D)质粒作为载体，用NotI- HF(NEB，R3189S)酶切开口，酶切的载体(8147bp)用Monarch DNA Gel Extraction Kit (NEB,T1020L)试剂盒回收。将5666F (CCAGAATCAGGGAAACGCGGCCGGATCCAAAAGAACTGCGGATGGATCTGAGTT TGAGA)(SEQ ID NO:32)和 5666R(GAAGTTAGTAGCGGATCCGACCTTCCTCTTCTTTTTTGGACTCTCAAACTCAG ATCCAT)(SEQ ID NO:33)引物对稀释到10μM，使用诺唯赞高保真酶试剂盒(Vazyme, p501-d2)引物之间互为模板pcr，PCR扩增产物经通过AxyPrep PCR Clean-up试剂盒 (Axygen，AP-PCR-500G)纯化回收后。用上述两个片段(酶切的片段、没有模板的PCR扩增片段)用
Figure BDA0002611487570000242
HiFi DNA Assembly Master Mix(E2621L)重组连接。室温孵育5min后转化涂板，经Sanger测序得到editor的C端添加有bpNLS质粒(CURE-N)，TC1332： CMVbpNLS-dPspCas13b-Apobec3A(Y132D)-bpNLS序列(SEQ ID NO:34)。
将4275Fb(GCGGCCGCTGGATCCGCTACTAACTTCAGCC)(SEQ ID NO:35)和4184R
(TCTTCTTGGGGCTCTCAAATTCGCTGCCGTCAGCAGTCCGTTTCATGGTGGCAAGCTTAAGTTTAAACGCTA)(SEQ ID NO:36)引物对稀释到10μM，以TC1309为载体使用诺唯赞高保真酶试剂盒(Vazyme，p501-d2)扩增出载体片段，将 4183F(AGAGCCCCAAGAAGAAGAGGAAAGTCGGATCCATCGAGAAGAAGAAGAGC TTCGCCAA)(SEQ ID NO:37)和4915R(CTTGCTGGTACCGTTGGTTCCCAGGATCCG) (SEQ ID NO:38)引物对稀释到10μM，以TC1316为模板，使用诺唯赞高保真酶试剂盒(Vazyme，p501-d2)扩增出所述第一dCas13Rx片段(核苷酸序列如SEQ ID NO:88 所示，氨基酸序列如SEQ ID NO:3所示)，将4916F (GAACCAACGGTACCAGCAAGAGGACAGCAGACGGGTCCGAATTCGAATCCCCCA AGAAA)(SEQ ID NO:39)和 4916R(CTGGCTTCCATAGACCCGGGACCCACCTTCCTCTTTTTCTTGGGGGATTCGAA TTCGGA)(SEQ ID NO:40)引物对稀释到10μM，使用诺唯赞高保真酶试剂盒(Vazyme, p501-d2)引物之间互为模板pcr扩增出第二bpNLS片段，将 4915F(GGGTCTATGGAAGCCAGCCCAGCATCCG)(SEQ ID NO:41)和4724F (CGCAGTTCTTTTGGATCCGTTTCCCTGATTCTGGAGAATG)(SEQ ID NO:42)引物对稀释到10μM，以TC1309为模板，使用诺唯赞高保真酶试剂盒(Vazyme，p501-d2) 扩增出Apobec3A(Y132D)片段，将4874F(GGATCCAAAAGAACTGCGGATGGATCT) (SEQ ID NO:43)和 4513R(GATGAAGTTGCGCATAGAACCGACCTTCCTCTTCTTTTTTGGAC)(SEQ ID NO:44)引物对稀释到10μM，以TC1316(SEQ ID NO:88)为模板，使用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)pcr扩增出第三bpNLS片段(SEQID NO:98)，将 4514(ATGCGCAACTTCATCATCAACAACG)(SEQ ID NO:45)和4183R (GGGGATTCGAACTCGGATCCATCGGCGGTGCGCTTAGAACCGGAGTTGCCGCTCA CCT)(SEQ ID NO:46)引物对稀释到10μM，以TC1316(SEQ ID NO:88)为模板，使用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)扩增出所述第二dCas13Rx片段(核苷酸序列如SEQ ID NO:97所示，氨基酸序列如SEQ ID NO:4所示)，将4858F (TGGATCCGAGTTCGAATCCCCTAAAAAGAAAAGAAAGGTGG)(SEQ ID NO:47) 和5802R(AGTAGCGGATCCAGCGGCCGCGGATCCCACCTTTCTTTTCTTTT)(SEQ ID NO:48)引物对稀释到10μM，使用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)引物之间互为模板pcr扩增出第四bpNLS片段。PCR扩增产物经通过AxyPrep PCR Clean-up试剂盒(Axygen,AP-PCR-500G)纯化回收后，上述片段用
Figure BDA0002611487570000261
HiFi DNA Assembly Master Mix(E2621L)重组连接。室温孵育15min后转化涂板，经Sanger测序得到CURE- X质粒TC1614:CMV-BPNLS-dCas13Rx(1I-558G)-BPNLS-Apobec3A(Y132D)-BPNLS- dCas13Rx(587M-966S)-BPNLS序列(SEQ ID NO:100)。
U6-BbsI-BbsI-PspCas13b DR crRNA质粒序列如下所示(SEQ ID NO:49)。根据靶向位点序列分别设计特异结构的碱基互补配对的上下游引物，加灭菌水溶解至100μM。经退火后连接到U6-BbsI-BbsI-PspCas13b DR crRNA载体上，以构建靶向特异性sgRNA。 sgRNA的引物序列如下：
6492T_PGAP2 accgTAAGTGAGCACTAGGAGGTTCTCCACGA(Spacer28,Loop6) (SEQ IDNO:50)
6492B_PGAP2 caacTCGTGGAGAACCTCCTAGTGCTCACTTA(Spacer28,Loop6) (SEQ IDNO:51)
6493T_PGAP2 accgCATAAGTGAGCACTAGGAGGTTCTCCACGACA (Spacer32,Loop6)(SEQID NO:52)
6493B_PGAP2 caacTGTCGTGGAGAACCTCCTAGTGCTCACTTATG (Spacer32,Loop6)(SEQID NO:53)
6494T_PGAP2 accgAGGAGACATAAGTGAGCACTAGGAGGTTCTCCACGACATTGAGG(Spacer44,Loop6)(SEQ ID NO:54)
6494B_PGAP2 caacCCTCAATGTCGTGGAGAACCTCCTAGTGCTCACTTATGTCTCCT(Spacer44,Loop6) (SEQ ID NO:55)
6495T_PGAP2 accgCGGAGGAGGAGACATAAGTGAGCACTAGGAGGTTCTCCACGACATTGAGGCCGAAG(Spacer56,Loop6)(SEQ ID NO:56)
6495B_PGAP2 caacCTTCGGCCTCAATGTCGTGGAGAACCTCCTAGTGCTCACTTATGTCTCCTCCTCC G(Spacer56,Loop6)(SEQ ID NO:57)
6488T_PGAP2 accgCATAAGTGAGCACTGGTTCTCCACGACA(Spacer28,Loop10) (SEQ IDNO:58)
6488B_PGAP2 caacTCGTGGAGAACCTCCTAGTGCTCACTTA(Spacer28,Loop10) (SEQ IDNO:59)
6489T_PGAP2 accgGACATAAGTGAGCACTGGTTCTCCACGACATT (Spacer32,Loop10)(SEQ ID NO:60)
6489B_PGAP2 caacAATGTCGTGGAGAACCAGTGCTCACTTATGTC (Spacer32,Loop10)(SEQ ID NO:61)
6490T_PGAP2 accgGGAGGAGACATAAGTGAGCACTGGTTCTCCACGACATTGAGGCC(Spacer44,Loop10)(SEQ ID NO:62)
6490B_PGAP2 caacGGCCTCAATGTCGTGGAGAACCAGTGCTCACTTATGTCTCCTCC(Spacer44,Loop10)(SEQ ID NO:63)
6491T_PGAP2 accgCTCGGAGGAGGAGACATAAGTGAGCACTGGTTCTCCACGACATTGAGGCCGAAGTT(Spacer56,Loop10)(SEQ ID NO:64)
6491B_PGAP2 caacAACTTCGGCCTCAATGTCGTGGAGAACCAGTGCTCACTTATGTCTCCTCCTCCG AG(Spacer56,Loop10)(SEQ ID NO:65)
6496T_PGAP2 accgGACATAAGTGAGCATTCTCCACGACATT(Spacer28,Loop14) (SEQ IDNO:66)
6496B_PGAP2 caacAATGTCGTGGAGAATGCTCACTTATGTC(Spacer28,Loop14) (SEQ IDNO:67)
6497T_PGAP2 accgGAGACATAAGTGAGCATTCTCCACGACATTGA (Spacer32,Loop14)(SEQ ID NO:68)
6497B_PGAP2 caacTCAATGTCGTGGAGAATGCTCACTTATGTCTC (Spacer32,Loop14)(SEQ ID NO:69)
6498T_PGAP2 accgGAGGAGGAGACATAAGTGAGCATTCTCCACGACATTGAGGCCGA(Spacer44,Loo p14)(SEQ ID NO:70)
6498B_PGAP2 caacTCGGCCTCAATGTCGTGGAGAATGCTCACTTATGTCTCCTCCTC(Spacer44,Loop14)(SEQ ID NO:71)
6499T_PGAP2 accgTCCTCGGAGGAGGAGACATAAGTGAGCATTCTCCACGACATTGAGGCCGAAGTTGA(Spacer56,Loop14)(SEQ ID NO:72)
6499B_PGAP2 caacTCAACTTCGGCCTCAATGTCGTGGAGAATGCTCACTTATGTCTCCTCCTCCGAG GA(Spacer56,Loop14)(SEQ ID NO:73)
利用BbsI-HF(NEB,R3539L)对U6-BbsI-BbsI-PspCas13b DR crRNA质粒进行酶切以得到线性化sgRNA载体。酶切的载体用Monarch DNA Gel Extraction Kit(NEB,T1020L)试剂盒回收得到线性化载体。取10ng线性化载体与0.3μl退火产物通过T4 DNA Ligase(NES，M0202S)连接，室温孵育30分钟后并转化涂板，经Sanger测序得到正确的靶向特异性sgRNA。
U6-Cas13Rx crRNA-BspQI-BspQI-Cas13Rx crRNA质粒合成于金斯瑞公司，序列(SEQ ID NO:74)根据靶向位点序列分别设计特异结构碱基互补配对的上下游引物，加灭菌水溶解至100μM。经退火后连接到U6-Cas13Rx crRNA-BspQI-BspQI-Cas13Rx crRNA载体上，以构建靶向特异性sgRNA。sgRNA的引物序列如下：
8101T_PGAP2 aacCATAAGTGAGCACTAGGAGGTTCTCCACGACA (Spacer32,Loop6)(SEQID NO:75)
8101B_PGAP2 ttgTGTCGTGGAGAACCTCCTAGTGCTCACTTATG (Spacer32,Loop6)(SEQID NO:76)
8102T_PGAP2 aacGACATAAGTGAGCACTGGTTCTCCACGACATT (Spacer32,Loop10)(SEQID NO:77)
8102B_PGAP2 ttgAATGTCGTGGAGAACCAGTGCTCACTTATGTC (Spacer32,Loop10)(SEQID NO:78)
8103T_PGAP2 aacGAGACATAAGTGAGCATTCTCCACGACATTGA (Spacer32,Loop14)(SEQID NO:79)
8103B_PGAP2 ttgTCAATGTCGTGGAGAATGCTCACTTATGTCTC (Spacer32,Loop14)(SEQID NO:80)
8118T_PGAP2 aacGACATAAGTGAGCATTCTCCACGACATT(Spacer28,Loop14) (SEQ IDNO:81)
8118B_PGAP2 ttgAATGTCGTGGAGAATGCTCACTTATGTC(Spacer28,Loop14) (SEQ IDNO:82)
利用BspQI(NEB,R0712L)对U6-Cas13Rx crRNA-BspQI-BspQI-Cas13Rx crRNA质粒进行酶切以得到线性化sgRNA载体。酶切的载体用Monarch DNA Gel Extraction Kit(NEB,T1020L)试剂盒回收得到线性化载体。取10ng线性化载体与0.3μl退火产物通过T4DNA Ligase(NES，M0202S)连接，室温孵育30分钟后并转化涂板，经Sanger测序得到正确的靶向特异性sgRNA。
实施例2
CURE系统在报告系统上的碱基编辑效率比较：
利用上述的CURE系统转染293T细胞，过程如下。
报告基因的操作步骤如下：
1)293T细胞(来自ATCC)复苏，在10cm培养皿(Falcon,353003)中培养，培养基为混有10％的胎牛血清(HyClone,SV30087)的DMEM(Gibco,C11995500BT)。培养温度为37℃，二氧化碳浓度为5％。多次传代后当细胞密度为80％时，细胞分盘至24孔板。
2)当细胞浓度为70-80％时，用10％血清的DMEM培养基换液，培养2小时使细胞状态恢复最佳。每孔转染的质粒的量分别是CURE质粒(CURE-C1、CURE-C2、CURE- X、CURE-N)300ng，sgRNA质粒200ng，荧光报告系统包含的两个质粒(序列分别如 SEQ ID NO:89和SEQID NO:101所示)各25ng。将质粒与在25μl混有0.75ul Lipofectamine P3000(ThermoFisher Scientific,L3000015)的Opti-MEM(Gibco,51985034) 培养基混匀。
3)将0.75μl Lipofectamine 3000(Thermo Fisher Scientific,L3000015)与25μl的Opti- MEM培养基混匀，静置5分钟。
4)将混有与P3000混好质粒的培养基加入混有Lipofectamine 3000的Opti-MEM，慢速吹打混匀，静置15分钟。
5)将混有质粒和转染试剂的Opti-MEM分别加入48孔板。
6)转染6小时后用10％FBS的DMEM补液。
7)转染16小时后，用10％FBS的DMEM换液。
8)转染48小时后收细胞，按照文献的方法用预冷的PBS洗去多余的DMEM，加配好的裂解液，裂出mRNA(Joung et al.,2017)。如果需要流式分析，则用预冷的PBS洗去多余的DMEM后加胰酶(life,25200072)消化,再用完全培养基DMEM终止消化。一半的细胞用于流式分析，一半的细胞离心沉淀去上清用PBS清洗两遍去上清再加裂解液，裂出mRNA。2μl裂解液用诺唯赞逆转录试剂盒HiScript II Q RT SuperMix kit(Vazyme， R223-01)进行逆转录，转录体系为10μl。
9)设计并合成PCR引物，这里GFP报告基因(图4、5、7、8、9、10、12、22、 23)的引物为(GCCACAAGCTGGAGTACAAC(SEQ ID NO:83)和 TAGAGAACCTCCCTGTCAGGTA)(SEQ ID NO:84)，加水稀释至10μM。用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)PCR扩增靶向位点片段。PCR产物样品用AxyPrep DNA 凝胶回收试剂盒(Axygen,AP-GX-250G)做割胶回收，去除非特异性条带；送deep-seq 测序分析。
内源基因的操作步骤：
1)293T细胞(来自ATCC)复苏，在10cm培养皿(Falcon,353003)中培养，培养基为混有10％的胎牛血清(HyClone,SV30087)的DMEM(Gibco,C11995500BT)。培养温度为37℃，二氧化碳浓度为5％。多次传代后当细胞密度为80％时，细胞分盘至24孔板。
2)当细胞浓度为70-80％时，用10％血清的DMEM培养基换液，培养2小时使细胞状态恢复最佳。每孔转染的质粒的量分别是CURE质粒(CURE-C1、CURE-C2、CURE- X、CURE-N)600ng，sgRNA质粒400ng。将质粒与在50μl混有1.5ul Lipofectamine P3000 (ThermoFisher Scientific,L3000015)的Opti-MEM(Gibco,51985034)培养基混匀。
3)将1.5μl Lipofectamine 3000(Thermo Fisher Scientific,L3000015)与50μl的Opti- MEM培养基混匀，静置5分钟。
4)将混有与P3000混好质粒的培养基加入混有Lipofectamine 3000的Opti-MEM，慢速吹打混匀，静置15分钟。
5)将混有质粒和转染试剂的Opti-MEM分别加入24孔板。
6)转染6小时后用10％FBS的DMEM补液。
7)转染16小时后，用10％FBS的DMEM换液。
8)转染48小时后收细胞，用预冷的PBS洗去多余的DMEM后加胰酶(life,25200072)消化,再用完全培养基DMEM终止消化。分选仪器分选出全红色阳性的细胞(editor上有 P2A跟着的mcherry)，收到的细胞离心沉淀去上清，加好裂解液，裂出mRNA。2μl裂解液用诺唯赞逆转录试剂盒HiScript II Q RT SuperMix kit(Vazyme，R223-01)进行逆转录，转录体系为10μl。
9)设计并合成相关位点的PCR引物，加水稀释至10μM。用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)PCR扩增靶向位点片段。PCR产物样品用AxyPrep DNA凝胶回收试剂盒(Axygen,AP-GX-250G)做割胶回收，去除非特异性条带；送deep-seq测序分析。
全转录组随机脱靶情况：
全转录组随机脱靶情况对于RNA精准碱基编辑来说是重要的，关系到最终碱基编辑器是否能应用到体内。全转录组实验用到的碱基编辑器有CURE-C1、CURE-C2、CURE- X、CURE-N、能编辑RNA的野生型脱氨酶APOBEC 3A(SEQ ID No:85)以及Abudayyeh et al.,2019文献中报道的RESCUE-S。编辑器与GFP荧光报告系统的质粒共转，分别加入相应的sgRNA。通过分选筛去没有转染进质粒的细胞。NGS分析荧光报告系统上的编辑效率为编辑器各自的编辑效率，而荧光报告系统之外的转录靶点为编辑器在转录组上的脱靶。
所得结果如图1-15所示。其中：
图1为示意图，其中上图为CURE载体示意图，下图为APOBEC 3A经典的编辑结构。
图2为GFP荧光报告基因荧光报告系统设计图，根据文献(Tunable andreversible drug control of protein production via a self-excising degron,Chung et al.,2015；Rational Design of a GFP-Based Fluorogenic Caspase Reporterfor Imaging Apoptosis In Vivo Author links open overlay panel Tsz-LeungTo etal.,2016)改造成的符合CURE系统初筛sgRNA或者CURE 编辑器的系统。GFP有11个β折叠，包括GFP(β1-10)和GFP(β11)两个部分(质粒)。两个质粒的组成如图(上下两部分的序列分别如SEQ ID NO:89和SEQ ID NO:101所示)，两部分前后分别被锌指拉链锁住了，GFP不完成，不发光。根据文献的描述，只要至少有一个锌指拉链消失，那这两部分就会组成新的GFP，从而发光，而NLS是为了使不编辑的时候β1-10部分能进到核里，后面的degron使其发生降解反应，而β11则在细胞质里，其目的都是降低背景。当独特的指导RNA将GFP(β1-10)与NLS之间的编辑位点RNA发生局部环化，使CGA脱氨形成UGA终止密码子，后面的NLS、ZIP、degron都不会被翻译出来，从而与GFP(β11)结合，从而复亮。
图3为sgRNA诱导荧光报告基因转录子形成的环状结构，其中编辑的位点为UAUC中的C。
图4为CURE-C1在荧光报告基因上编辑效率的结果(操作步骤为本领域常规)，A 和B图为Facs结果，C图为sanger结果。图4的A图是任意序列的指导RNA，因为没有在这个位点上发生脱氨，但是又因为这个荧光系统有点背景，所以有微弱的绿色。B图是针对这个报告系统的指导RNA，脱氨以后GFP复亮，而且与A图的对比明显，说明这个系统是可行的，GFP荧光报告基因是能用来初筛的。C图NT对应A图，sanger图用EditR(https://moriaritylab.shinyapps.io/editr_v10/)算出是2％，但从sanger测序结果 ab1图来看几乎没有峰，所以可能为零，图5的NGS结果也证明编辑为零，而加了sgRNA 之后编辑效率高达40％，与上图的右图基本符合，更进一步说明荧光系统的实用性。
图5为CURE-C1在在荧光报告基因上编辑效率的NGS结果(操作步骤为本领域常规)，其中图A为on-target的sgRNA与其他sgRNA的结构图，图B为各个sgRNA编辑效率的deep-seq分析测序结果。由图中可知，只有dCas13b与A3A形成融合蛋白且在能将靶点局部环化的指导RNA(序列如图5的gRNA#1所示(SEQ ID NO:92))下才能够编辑(定点编辑)。
图6为CURE-C1编辑内源碱基编辑效率sanger测序结果与deep-seq测序分析结果比较(操作步骤为本领域常规)。由图中可知，这三个是编辑的内源基因，初试CURE在内源基因的编辑情况，sanger和NGS的结果表明，NGS的编辑效率与EditR算出sanger 的编辑效率比较吻合，因此后续可以先以sanger来判断编辑情况。
图7为CURE-C1和CURE-X在不同长度sgRNA引导下在报告系统上的编辑效率的测序结果。左图为sgRNA在报告基因上的位置，其中UAUC在状结构图的正中间；右图为编辑效率的测序结果，其中CURE-C1是sanger测序结果，CURE-X是deep-seq测序分析结果。由图中可知，在标准的(UAUC在中间)14个碱基环状的编辑框下，CURE- C1在指导RNA与靶位点是32个碱基的结合下的编辑效率比较高，达到40％左右，较短的指导RNA和长的指导RNA编辑效率呈下降趋势，是一代测序。而CURE-X最优的指导RNA是28个碱基，编辑效率为30％以上，再减四个碱基，下降明显，编辑效率仅为 10％，碱基数上升，编辑效率也呈下降趋势，44个碱基之后，基本不编辑了，是二代测序。从图可以得到的规律是对于编辑框有14个碱基环来说，CURE-C1(对CURE- C2,CURE-N也适用，因为它们三都是相同的RNA结合蛋白-dpspCasb)编辑某个基因时，可以选择28个碱基与36个碱基数的指导RNA，优先选择32个碱基的指导RNA。同理 CURE-X选择指导RNA时，可以选择28个碱基以及周围的几个碱基数。
图8为CURE-C1在长度为32nt的sgRNA引导下和CURE-X在长度为28nt的sgRNA 引导下报告系统形成不同长度的环状结构的编辑效率的测序结果。左图为不同长度的环状结构图，其中UAUC在状结构图的正中间；右图为编辑效率的测序结果，其中CURE- C1是sanger测序结果，CURE-X是deep-seq测序分析结果。由图中可知，在CURE-C1 的指导RNA为32个碱基情况下，CURE-X的指导RNA为28个碱基的情况下，去探索由指导RNA将靶点RNA局部环化形成编辑框里的碱基个数。实验发现CURE-C1除了编辑框有4个碱基时编辑效率是15％，比较低以外，从6-20个碱基的编辑效率高达40- 60％，是一代测序，而CURE-X是8、12、14个碱基时编辑效率高达40％以上，10、16、 18、20个碱基时编辑效率较高是20-30％，4和6个碱基几乎不编辑。从图上可知对于 CURE-C1(对CURE-C2,CURE-N也适用，因为它们三都是相同的RNA结合蛋白-dpspCasb) 来说，编辑框里碱基范围的选择比较广6-20个碱基内的可以选择，也可以选择比20个碱基还多的条件，而CURE-X的的选择的范围要窄一些，建议是10-14个碱基数内选择。
图9为CURE-C1在长度为32nt的sgRNA引导下和CURE-X在长度为28nt的sgRNA 引导报告系统形成14nt环状结构的下编辑UC在环状结构里的不同位置的编辑效率的测序结果。左图UAUC编辑位点的位置，右图为编辑效率的测序结果，其中CURE-C1是sanger测序结果，CURE-X是deep-seq测序分析结果。图9中继续探索了编辑框里UAUC 的位置除了放中间还能否放其他位置，其目的还是探索一下指导RNA设计位置的可能性。根据图7和图8的实验结果把指导RNA的长度固定在CURE-C1是32个碱基， CURE-X是28个碱基，编辑框两种编辑系统都固定为14个碱基环。UAUC中的C从编辑框里的第5个碱基一直一刀第11个碱基。从结果上看，CURE-C1系统的容忍度很高，编辑效率几乎都很高，高达40％以上，除了放在第10位C，但编辑效率也达到了30％。而CURE-X编辑不了放在第5位的C，其余位置编辑效率差不多，有20％以上。这个实验结果说明C的位置还是相对有包容性的，除了CURE-X系统里设计指导RNA时注意不要把C放到第5位之外，都可以以根据实验的需求比较随意的设计指导RNA。
图10为CURE-C1在长度为32nt的sgRNA引导下和CURE-X在长度为28nt的 sgRNA引导报告系统形成14nt环状结构的下的测序结果。左图UC编辑位点两边不同碱基的位置，右图为编辑效率的测序结果，其中CURE-C1是sanger测序结果，CURE-X是 deep-seq测序分析结果。之前都是把UAUCG放一块来进行编辑，图10中探索了C周围的序列对C编辑的影响。NNCN，每个N分别以A,T,C,G来依次代替AUCG，从图上来看CURE-C1和CURE-X的结果一致，这是由APOBEC 3A本身能编辑的基序决定的。 CURE只能编辑C前面为U；C后面的碱基为U或者C时，编辑效率会下降(20％)，而为G或A时编辑效率高达40％以上；UC前面一个序列也是对C的编辑有影响，为A 时编辑效率是40％以上，是G或U或C时是25％左右。可以根据不同的基序选择合适的编辑位点，达到编辑系统的最优化。
图11a为不同sgRNA引导下外源基因形成不同环状结构的结构图，其中同个环里可能包含两个编辑UC位点，这里暂把右边的UC定为实际编辑的位点，而左边的UC为额外编辑的位点。实验中在修复点突变造成疾病的序列时，发现CURE能同时编辑编辑框里的UC，因此做了很多指导RNA，发现指导RNA的编辑灵活性很好。
图11b为CURE-C1在图11a上的编辑效率结果，左图为sanger分析结果图，右图为左图其中几个值的sanger测序结果ab1图。根据之前在报告系统上探索的指导RNA设计了比较密集针对这个点突变疾病位点一系列的指导RNA，发现通过缩小编辑框里的碱基环，缩小到6个碱基，把不需要编辑的位点排除之外，或者把它安排到旁边(比如10 个碱基环的时候)就有效的防止额外的编辑。但如果是想要同时编辑两个UC时，可以选择增加编辑框里碱基环的个数，如增加到14个碱基，通过指导RNA的设计把两个UC 都放在了较合适，再选择合适长度的指导RNA(如图7，对于CURE-C1来说14个碱基环的编辑框，32个碱基长度的指导RNA是最合适的)，如图中32个碱基数的指导RNA，两个UC都被编辑的效率就会很高，都大于40％。
图11c为CURE在图11a上的deep-seq测序分析结果，上图为CURE-C1分析结果图，右图CURE-X分析结果图。根据之前的实验结果，进一步通过二代测序观察两个编辑UC位点的编辑情况，选择指导RNA长度为32，编辑框里的碱基分别为6个，10个和14个，而CURE-X多做了指导RNA为28个碱基，编辑框里碱基数为14的指导RNA。从结果上看，虽然CURE-X同时编辑两个UC的能力不佳，但是CURE-C1是很出众的，而且几乎两个UC是同时编辑的，发生在同一个mRNA上，说明CURE-C1的编辑能力很强，能同时编辑掉同一个编辑框里两个编辑位点，也可能存在编辑更多存在于编辑框里多个编辑位点的潜能。
图12为CURE与RESCUE-S在外源基因上靶向UCC的编辑效率，其中右图为CURE 和RESCUE-S的sgRNA分别在UCC外源基因上的位置；左图为deep-seq测序分析结果。由图中可以看出，CURE能将UCC一步编辑变为UUU，即能把脯氨酸直接变成苯丙氨酸或者亮氨酸，这是RESCUE-S所不能做到的。
图13为CURE与RESCUE-S在内源转录本上靶向UC的编辑效率的deep-seq测序分析结果比较，其中高亮的基因为RESCUE-S文章里的位点。由图中可以看出，碱基编辑器CURE-C2在PPIB(I18I)、TYMS(I262I)、ACTB(I75I)基因上的编辑效率高达40％- 60％，而现有的RESCUE-S在这几个基因上的编辑效率为10％-40％。
图14为CURE与RESCUE-S-N在内源靶向细胞核内转录本UC的编辑效率的deep- seq测序分析结果比较，其中RESCUE-S-N是RESCUE-S核定位版本。在核内编辑的 CURE版本CURE-N能较高效率的编辑只存在于细胞核里的RNA，进一步尝试把 RESCURE-S也加上入核信号，把RESCUE-S也拉进核里进行编辑，发现对于MALAT1 和XIST这两个位点来说，编辑效率远不如CURE-N的编辑。所以对于只存在于核内的 RNA C>U编辑来说，CURE-N的优势相当明显。
图15为CURE和RESCUE-S在荧光报告系统中在sgRNA引导时的全转录组脱靶情况，其中CURE-C2和CURE-N的sgRNA是32-14，CURE-X的sgRNA是28-14，RESCUE- S的sgRNA是30/22(转染按照本领域常规的操作即可，按48孔板的报告基因的条件放大到24孔板，每个参数x4。分选也使用的本领域常规操作。用trizol法提RNA；并将 RNA送到测序公司，由公司来建库测序)。从图15上看，各编辑器在荧光系统上的编辑效率是正常的。A3A它是识别并结合特殊的环状结构的脱氨酶，而荧光报告系统没有特殊结构，所以A3A编辑器没有编辑。CUREs及A3A脱氨C>U编辑器在转录组上没有 A>I的脱氨活性，而RESCUE-S显示有860个脱靶位点。CURE-C2在转录组上有7416 个，比RESCUE-S脱靶位点(1471)高了5倍。之后优化了的CURE版本，换了Cas13Rx 的结合蛋白，CURE-X的脱靶位点大大下降，相比起最初的版本CURE-C2的脱靶位点个数下降了将近20倍，从7416个(CURE-C2)下降到了380个(CURE-X)。CURE-X (380个)相比起RESCUE-S(C>U 611个，A>I 860个)的特异性也明显提高，但由于 CURE-X在某些内源基因上的编辑效率不是很理想，所以新的版本CURE-N在内源基因的编辑与RESCUE-S相当，脱靶的实际个数也相当。
图16和17为使用了有其他的胞苷脱氨酶APOBEC 3G(简称A3G，Gene ID:60489)、APOBEC 1(简称A1，Gene ID:339)、mAPOBEC 1(简称mA1，Gene ID:11810)、 APOBEC 2(简称A2，GeneID:10930)应用于本申请的实验中的结果(实验步骤同图7，仅靶基因不同)，但均不能实现C>U的编辑。
图18为包含不同突变体的APOBEC 3A片段的实验结果图(实验步骤同图7)，由图中可知，仅包含突变Y132D和H29K的APOBEC 3A片段的效果较好。
图19a为Abudayyeh et al.,2019里的原图。图19为根据Abudayyeh et al.,2019里的原图分析之后算出来的实验数据整合成的表格。图20为根据Abudayyeh et al.,2019里的原图分析之后算出来的实验数据整合成的表格。结果表明，RESCUE在对内源性转录本的编辑中，对靶位点C前面的碱基有偏好性，更倾向于编辑AC和UC，G/CC(CC和 GC)序列编辑效率较低，而因为RESCUE-S活性通常不如RESCUE，这种偏好现象在 RESCUE-S中更为严重。事实上，RESCUE-S对内源性转录本中G/CC序列的编辑效率通常低于2％，这使得它实际上不实用。最后，虽然RESCUE-S可以编辑AC和UC，但UC编辑的效率较低，甚至在靶位点UC的编辑率低于4％。
图22中显示了尝试使用其他RNA结合蛋白MCP(MS2 Coat Protein，靶向RNA 的结合蛋白)和CIRTS(CRISPR-Cas-inspired RNA targeting system，靶向RNA的结合蛋白)应用于本申请的实验中的结果(Sanger测序，实验步骤同图7)，结果是阴性的，均不能实现C>U的编辑。
图23中显示了将dCas13Rx置于APOBEC 3A的C端或其他位置时的结果图 (Deep-seq测序)。由图中可以看出，将dCas13Rx置于APOBEC 3A的C端或其他位置时，同样有较好的编辑效率。
可见，CURE-X是目前为止最特异的C>U碱基编辑器，而CURE-N与RESCUE-S碱基编辑器相当，但是比RESCUE-S在编辑位点上的编辑方式或效应有优势(比如CURE- N能编辑只存在于细胞核里的RNA，以及CURE-N能把UCC一步编辑成UUU，这是 RESCUE-S做不到的)。这证明CUREs系统是目前基于CRISPR系统的RNA碱基编辑中不可或缺系统。
基因组上的脱靶情况：
如图21，David Liu团队在今年2月份发表了(Evaluation and minimization ofCas9- independent off-target DNA editing by cytosine base editors Doman etal.,2020)通过放大 DNA脱靶位点的信号能进行编辑器在基因组上脱靶位点的检测。通过另外一种类型的 dsaCas9在指导RNA的带领下把基因组上C>U容易脱靶的位点打开并固定，使C>U的编辑器更容易靠近，对于David Liu的编辑系统来说是DNA C>U的编辑器BE4，对于本申请来讲，是本靶向与RNA的，看是否会同时靶向DNA的CURE编辑系统。从实验结果上看(转染质粒的总量为：Editor(CURE/BE4):600ng；Editor sgRNA:400ng；dSa Cas9(结合DNA序列，起到在基因组增强胞苷酶在这位点上胞苷脱氨的信号)：600ng；dSa Cas9 sgRNA:400ng；转染的试剂，P3000和lipo3000转24孔板的量X1.5。分选后，基因组需要用DirectPCRLysis Reagent(cell)(viagen，301-C)加上适量的PK，55℃裂解过夜再 PCR)，本申请的实施例用的CURE在二代测序下R-loop(基因组上容易脱靶的位点) 显示的编辑效率均为1,2或3，而阴性对照(NC无编辑器)的效率也为1,2或3，所以相当于没有发生这个基因组脱靶效应。而在CURE-N后面加了和BE4max一样的2X UGI 了之后，我们发现它在这个位点有了不明显的脱靶效应，说明CURE-N的确有在基因组发生脱氨效应，但由于机体内有DNA上U去除的修复(UGI是阻止这个修复的)的功能也就是说在没有UGI的情况下，CURE-N编辑了少量的DNA之后会被机体修复回去。
综上所述，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
SEQUENCE LISTING
<110> 上海科技大学
<120> 一种用于C到U碱基编辑的融合蛋白及其制备方法和应用
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<170> PatentIn version 3.5
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Ala Ser Arg Thr Glu Arg Tyr Arg Lys Gln Ala Ser Asn Lys Ile Arg
835 840 845
Ser Asn Arg Gln Met Arg Asn Ala Ser Ser Glu Glu Ile Glu Thr Ile
850 855 860
Leu Asp Lys Arg Leu Ser Asn Ser Arg Asn Glu Tyr Gln Lys Ser Glu
865 870 875 880
Lys Val Ile Arg Arg Tyr Arg Val Gln Asp Ala Leu Leu Phe Leu Leu
885 890 895
Ala Lys Lys Thr Leu Thr Glu Leu Ala Asp Phe Asp Gly Glu Arg Phe
900 905 910
Lys Leu Lys Glu Ile Met Pro Asp Ala Glu Lys Gly Ile Leu Ser Glu
915 920 925
Ile Met Pro Met Ser Phe Thr Phe Glu Lys Gly Gly Lys Lys Tyr Thr
930 935 940
Ile Thr Ser Glu Gly Met Lys Leu Lys Asn Tyr Gly Asp Phe Phe Val
945 950 955 960
Leu Ala Ser Asp Lys Arg Ile Gly Asn Leu Leu Glu Leu Val Gly Ser
965 970 975
Asp Ile Val Ser Lys Glu Asp
980
<210> 2
<211> 199
<212> PRT
<213> Artificial Sequence
<220>
<223> APOBEC3A(Y132D)片段
<400> 2
Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His
1 5 10 15
Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr
20 25 30
Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met
35 40 45
Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys
50 55 60
Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro
65 70 75 80
Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile
85 90 95
Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala
100 105 110
Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg
115 120 125
Ile Tyr Asp Asp Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg
130 135 140
Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His
145 150 155 160
Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp
165 170 175
Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala
180 185 190
Ile Leu Gln Asn Gln Gly Asn
195
<210> 3
<211> 558
<212> PRT
<213> Ruminococcus flavefaciensstrain XPD3002
<400> 3
Ile Glu Lys Lys Lys Ser Phe Ala Lys Gly Met Gly Val Lys Ser Thr
1 5 10 15
Leu Val Ser Gly Ser Lys Val Tyr Met Thr Thr Phe Ala Glu Gly Ser
20 25 30
Asp Ala Arg Leu Glu Lys Ile Val Glu Gly Asp Ser Ile Arg Ser Val
35 40 45
Asn Glu Gly Glu Ala Phe Ser Ala Glu Met Ala Asp Lys Asn Ala Gly
50 55 60
Tyr Lys Ile Gly Asn Ala Lys Phe Ser His Pro Lys Gly Tyr Ala Val
65 70 75 80
Val Ala Asn Asn Pro Leu Tyr Thr Gly Pro Val Gln Gln Asp Met Leu
85 90 95
Gly Leu Lys Glu Thr Leu Glu Lys Arg Tyr Phe Gly Glu Ser Ala Asp
100 105 110
Gly Asn Asp Asn Ile Cys Ile Gln Val Ile His Asn Ile Leu Asp Ile
115 120 125
Glu Lys Ile Leu Ala Glu Tyr Ile Thr Asn Ala Ala Tyr Ala Val Asn
130 135 140
Asn Ile Ser Gly Leu Asp Lys Asp Ile Ile Gly Phe Gly Lys Phe Ser
145 150 155 160
Thr Val Tyr Thr Tyr Asp Glu Phe Lys Asp Pro Glu His His Arg Ala
165 170 175
Ala Phe Asn Asn Asn Asp Lys Leu Ile Asn Ala Ile Lys Ala Gln Tyr
180 185 190
Asp Glu Phe Asp Asn Phe Leu Asp Asn Pro Arg Leu Gly Tyr Phe Gly
195 200 205
Gln Ala Phe Phe Ser Lys Glu Gly Arg Asn Tyr Ile Ile Asn Tyr Gly
210 215 220
Asn Glu Cys Tyr Asp Ile Leu Ala Leu Leu Ser Gly Leu Ala His Trp
225 230 235 240
Val Val Ala Asn Asn Glu Glu Glu Ser Arg Ile Ser Arg Thr Trp Leu
245 250 255
Tyr Asn Leu Asp Lys Asn Leu Asp Asn Glu Tyr Ile Ser Thr Leu Asn
260 265 270
Tyr Leu Tyr Asp Arg Ile Thr Asn Glu Leu Thr Asn Ser Phe Ser Lys
275 280 285
Asn Ser Ala Ala Asn Val Asn Tyr Ile Ala Glu Thr Leu Gly Ile Asn
290 295 300
Pro Ala Glu Phe Ala Glu Gln Tyr Phe Arg Phe Ser Ile Met Lys Glu
305 310 315 320
Gln Lys Asn Leu Gly Phe Asn Ile Thr Lys Leu Arg Glu Val Met Leu
325 330 335
Asp Arg Lys Asp Met Ser Glu Ile Arg Lys Asn His Lys Val Phe Asp
340 345 350
Ser Ile Arg Thr Lys Val Tyr Thr Met Met Asp Phe Val Ile Tyr Arg
355 360 365
Tyr Tyr Ile Glu Glu Asp Ala Lys Val Ala Ala Ala Asn Lys Ser Leu
370 375 380
Pro Asp Asn Glu Lys Ser Leu Ser Glu Lys Asp Ile Phe Val Ile Asn
385 390 395 400
Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys Asp Ala Leu Tyr Tyr Asp
405 410 415
Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu Asn Ile Met His Asn Ile
420 425 430
Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu Tyr Lys Lys Lys Asp Ala
435 440 445
Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly Arg Asp Val Ser Ala Phe
450 455 460
Ser Lys Leu Met Tyr Ala Leu Thr Met Phe Leu Asp Gly Lys Glu Ile
465 470 475 480
Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe Asp Asn Ile Gln Ser
485 490 495
Phe Leu Lys Val Met Pro Leu Ile Gly Val Asn Ala Lys Phe Val Glu
500 505 510
Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys Ile Ala Asp Glu Leu Arg
515 520 525
Leu Ile Lys Ser Phe Ala Arg Met Gly Glu Pro Ile Ala Asp Ala Arg
530 535 540
Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile Leu Gly Thr Asn
545 550 555
<210> 4
<211> 380
<212> PRT
<213> Ruminococcus flavefaciensstrain XPD3002
<400> 4
Met Arg Asn Phe Ile Ile Asn Asn Val Ile Ser Asn Lys Arg Phe His
1 5 10 15
Tyr Leu Ile Arg Tyr Gly Asp Pro Ala His Leu His Glu Ile Ala Lys
20 25 30
Asn Glu Ala Val Val Lys Phe Val Leu Gly Arg Ile Ala Asp Ile Gln
35 40 45
Lys Lys Gln Gly Gln Asn Gly Lys Asn Gln Ile Asp Arg Tyr Tyr Glu
50 55 60
Thr Cys Ile Gly Lys Asp Lys Gly Lys Ser Val Ser Glu Lys Val Asp
65 70 75 80
Ala Leu Thr Lys Ile Ile Thr Gly Met Asn Tyr Asp Gln Phe Asp Lys
85 90 95
Lys Arg Ser Val Ile Glu Asp Thr Gly Arg Glu Asn Ala Glu Arg Glu
100 105 110
Lys Phe Lys Lys Ile Ile Ser Leu Tyr Leu Thr Val Ile Tyr His Ile
115 120 125
Leu Lys Asn Ile Val Asn Ile Asn Ala Arg Tyr Val Ile Gly Phe His
130 135 140
Cys Val Glu Arg Asp Ala Gln Leu Tyr Lys Glu Lys Gly Tyr Asp Ile
145 150 155 160
Asn Leu Lys Lys Leu Glu Glu Lys Gly Phe Ser Ser Val Thr Lys Leu
165 170 175
Cys Ala Gly Ile Asp Glu Thr Ala Pro Asp Lys Arg Lys Asp Val Glu
180 185 190
Lys Glu Met Ala Glu Arg Ala Lys Glu Ser Ile Asp Ser Leu Glu Ser
195 200 205
Ala Asn Pro Lys Leu Tyr Ala Asn Tyr Ile Lys Tyr Ser Asp Glu Lys
210 215 220
Lys Ala Glu Glu Phe Thr Arg Gln Ile Asn Arg Glu Lys Ala Lys Thr
225 230 235 240
Ala Leu Asn Ala Tyr Leu Arg Asn Thr Lys Trp Asn Val Ile Ile Arg
245 250 255
Glu Asp Leu Leu Arg Ile Asp Asn Lys Thr Cys Thr Leu Phe Ala Asn
260 265 270
Lys Ala Val Ala Leu Glu Val Ala Arg Tyr Val His Ala Tyr Ile Asn
275 280 285
Asp Ile Ala Glu Val Asn Ser Tyr Phe Gln Leu Tyr His Tyr Ile Met
290 295 300
Gln Arg Ile Ile Met Asn Glu Arg Tyr Glu Lys Ser Ser Gly Lys Val
305 310 315 320
Ser Glu Tyr Phe Asp Ala Val Asn Asp Glu Lys Lys Tyr Asn Asp Arg
325 330 335
Leu Leu Lys Leu Leu Cys Val Pro Phe Gly Tyr Cys Ile Pro Arg Phe
340 345 350
Lys Asn Leu Ser Ile Glu Ala Leu Phe Asp Arg Asn Glu Ala Ala Lys
355 360 365
Phe Asp Lys Glu Lys Lys Lys Val Ser Gly Asn Ser
370 375 380
<210> 5
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> bpNLS
<400> 5
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val
<210> 6
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> mNLS
<400> 6
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Ala Ala Ala
1 5 10 15
Lys
<210> 7
<211> 1217
<212> PRT
<213> Artificial Sequence
<220>
<223> CURE-C1
<400> 7
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val Asn Ile Pro Ala Leu Val Glu Asn Gln Lys Lys Tyr Phe Gly
20 25 30
Thr Tyr Ser Val Met Ala Met Leu Asn Ala Gln Thr Val Leu Asp His
35 40 45
Ile Gln Lys Val Ala Asp Ile Glu Gly Glu Gln Asn Glu Asn Asn Glu
50 55 60
Asn Leu Trp Phe His Pro Val Met Ser His Leu Tyr Asn Ala Lys Asn
65 70 75 80
Gly Tyr Asp Lys Gln Pro Glu Lys Thr Met Phe Ile Ile Glu Arg Leu
85 90 95
Gln Ser Tyr Phe Pro Phe Leu Lys Ile Met Ala Glu Asn Gln Arg Glu
100 105 110
Tyr Ser Asn Gly Lys Tyr Lys Gln Asn Arg Val Glu Val Asn Ser Asn
115 120 125
Asp Ile Phe Glu Val Leu Lys Arg Ala Phe Gly Val Leu Lys Met Tyr
130 135 140
Arg Asp Leu Thr Asn Ala Tyr Lys Thr Tyr Glu Glu Lys Leu Asn Asp
145 150 155 160
Gly Cys Glu Phe Leu Thr Ser Thr Glu Gln Pro Leu Ser Gly Met Ile
165 170 175
Asn Asn Tyr Tyr Thr Val Ala Leu Arg Asn Met Asn Glu Arg Tyr Gly
180 185 190
Tyr Lys Thr Glu Asp Leu Ala Phe Ile Gln Asp Lys Arg Phe Lys Phe
195 200 205
Val Lys Asp Ala Tyr Gly Lys Lys Lys Ser Gln Val Asn Thr Gly Phe
210 215 220
Phe Leu Ser Leu Gln Asp Tyr Asn Gly Asp Thr Gln Lys Lys Leu His
225 230 235 240
Leu Ser Gly Val Gly Ile Ala Leu Leu Ile Cys Leu Phe Leu Asp Lys
245 250 255
Gln Tyr Ile Asn Ile Phe Leu Ser Arg Leu Pro Ile Phe Ser Ser Tyr
260 265 270
Asn Ala Gln Ser Glu Glu Arg Arg Ile Ile Ile Arg Ser Phe Gly Ile
275 280 285
Asn Ser Ile Lys Leu Pro Lys Asp Arg Ile His Ser Glu Lys Ser Asn
290 295 300
Lys Ser Val Ala Met Asp Met Leu Asn Glu Val Lys Arg Cys Pro Asp
305 310 315 320
Glu Leu Phe Thr Thr Leu Ser Ala Glu Lys Gln Ser Arg Phe Arg Ile
325 330 335
Ile Ser Asp Asp His Asn Glu Val Leu Met Lys Arg Ser Ser Asp Arg
340 345 350
Phe Val Pro Leu Leu Leu Gln Tyr Ile Asp Tyr Gly Lys Leu Phe Asp
355 360 365
His Ile Arg Phe His Val Asn Met Gly Lys Leu Arg Tyr Leu Leu Lys
370 375 380
Ala Asp Lys Thr Cys Ile Asp Gly Gln Thr Arg Val Arg Val Ile Glu
385 390 395 400
Gln Pro Leu Asn Gly Phe Gly Arg Leu Glu Glu Ala Glu Thr Met Arg
405 410 415
Lys Gln Glu Asn Gly Thr Phe Gly Asn Ser Gly Ile Arg Ile Arg Asp
420 425 430
Phe Glu Asn Met Lys Arg Asp Asp Ala Asn Pro Ala Asn Tyr Pro Tyr
435 440 445
Ile Val Asp Thr Tyr Thr His Tyr Ile Leu Glu Asn Asn Lys Val Glu
450 455 460
Met Phe Ile Asn Asp Lys Glu Asp Ser Ala Pro Leu Leu Pro Val Ile
465 470 475 480
Glu Asp Asp Arg Tyr Val Val Lys Thr Ile Pro Ser Cys Arg Met Ser
485 490 495
Thr Leu Glu Ile Pro Ala Met Ala Phe His Met Phe Leu Phe Gly Ser
500 505 510
Lys Lys Thr Glu Lys Leu Ile Val Asp Val His Asn Arg Tyr Lys Arg
515 520 525
Leu Phe Gln Ala Met Gln Lys Glu Glu Val Thr Ala Glu Asn Ile Ala
530 535 540
Ser Phe Gly Ile Ala Glu Ser Asp Leu Pro Gln Lys Ile Leu Asp Leu
545 550 555 560
Ile Ser Gly Asn Ala His Gly Lys Asp Val Asp Ala Phe Ile Arg Leu
565 570 575
Thr Val Asp Asp Met Leu Thr Asp Thr Glu Arg Arg Ile Lys Arg Phe
580 585 590
Lys Asp Asp Arg Lys Ser Ile Arg Ser Ala Asp Asn Lys Met Gly Lys
595 600 605
Arg Gly Phe Lys Gln Ile Ser Thr Gly Lys Leu Ala Asp Phe Leu Ala
610 615 620
Lys Asp Ile Val Leu Phe Gln Pro Ser Val Asn Asp Gly Glu Asn Lys
625 630 635 640
Ile Thr Gly Leu Asn Tyr Arg Ile Met Gln Ser Ala Ile Ala Val Tyr
645 650 655
Asp Ser Gly Asp Asp Tyr Glu Ala Lys Gln Gln Phe Lys Leu Met Phe
660 665 670
Glu Lys Ala Arg Leu Ile Gly Lys Gly Thr Thr Glu Pro His Pro Phe
675 680 685
Leu Tyr Lys Val Phe Ala Arg Ser Ile Pro Ala Asn Ala Val Glu Phe
690 695 700
Tyr Glu Arg Tyr Leu Ile Glu Arg Lys Phe Tyr Leu Thr Gly Leu Ser
705 710 715 720
Asn Glu Ile Lys Lys Gly Asn Arg Val Asp Val Pro Phe Ile Arg Arg
725 730 735
Asp Gln Asn Lys Trp Lys Thr Pro Ala Met Lys Thr Leu Gly Arg Ile
740 745 750
Tyr Ser Glu Asp Leu Pro Val Glu Leu Pro Arg Gln Met Phe Asp Asn
755 760 765
Glu Ile Lys Ser His Leu Lys Ser Leu Pro Gln Met Glu Gly Ile Asp
770 775 780
Phe Asn Asn Ala Asn Val Thr Tyr Leu Ile Ala Glu Tyr Met Lys Arg
785 790 795 800
Val Leu Asp Asp Asp Phe Gln Thr Phe Tyr Gln Trp Asn Arg Asn Tyr
805 810 815
Arg Tyr Met Asp Met Leu Lys Gly Glu Tyr Asp Arg Lys Gly Ser Leu
820 825 830
Gln His Cys Phe Thr Ser Val Glu Glu Arg Glu Gly Leu Trp Lys Glu
835 840 845
Arg Ala Ser Arg Thr Glu Arg Tyr Arg Lys Gln Ala Ser Asn Lys Ile
850 855 860
Arg Ser Asn Arg Gln Met Arg Asn Ala Ser Ser Glu Glu Ile Glu Thr
865 870 875 880
Ile Leu Asp Lys Arg Leu Ser Asn Ser Arg Asn Glu Tyr Gln Lys Ser
885 890 895
Glu Lys Val Ile Arg Arg Tyr Arg Val Gln Asp Ala Leu Leu Phe Leu
900 905 910
Leu Ala Lys Lys Thr Leu Thr Glu Leu Ala Asp Phe Asp Gly Glu Arg
915 920 925
Phe Lys Leu Lys Glu Ile Met Pro Asp Ala Glu Lys Gly Ile Leu Ser
930 935 940
Glu Ile Met Pro Met Ser Phe Thr Phe Glu Lys Gly Gly Lys Lys Tyr
945 950 955 960
Thr Ile Thr Ser Glu Gly Met Lys Leu Lys Asn Tyr Gly Asp Phe Phe
965 970 975
Val Leu Ala Ser Asp Lys Arg Ile Gly Asn Leu Leu Glu Leu Val Gly
980 985 990
Ser Asp Ile Val Ser Lys Glu Asp Gly Ser Leu Gln Leu Pro Pro Leu
995 1000 1005
Glu Arg Leu Thr Leu Gly Ser Gly Ser Ser Met Glu Ala Ser Pro
1010 1015 1020
Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser
1025 1030 1035
Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr
1040 1045 1050
Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln
1055 1060 1065
His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly
1070 1075 1080
Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro
1085 1090 1095
Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe
1100 1105 1110
Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val
1115 1120 1125
Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe
1130 1135 1140
Ala Ala Arg Ile Tyr Asp Asp Asp Pro Leu Tyr Lys Glu Ala Leu
1145 1150 1155
Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr
1160 1165 1170
Asp Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly
1175 1180 1185
Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala
1190 1195 1200
Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn
1205 1210 1215
<210> 8
<211> 1217
<212> PRT
<213> Artificial Sequence
<220>
<223> CURE-C2
<400> 8
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Ala Ala Ala
1 5 10 15
Lys Val Asn Ile Pro Ala Leu Val Glu Asn Gln Lys Lys Tyr Phe Gly
20 25 30
Thr Tyr Ser Val Met Ala Met Leu Asn Ala Gln Thr Val Leu Asp His
35 40 45
Ile Gln Lys Val Ala Asp Ile Glu Gly Glu Gln Asn Glu Asn Asn Glu
50 55 60
Asn Leu Trp Phe His Pro Val Met Ser His Leu Tyr Asn Ala Lys Asn
65 70 75 80
Gly Tyr Asp Lys Gln Pro Glu Lys Thr Met Phe Ile Ile Glu Arg Leu
85 90 95
Gln Ser Tyr Phe Pro Phe Leu Lys Ile Met Ala Glu Asn Gln Arg Glu
100 105 110
Tyr Ser Asn Gly Lys Tyr Lys Gln Asn Arg Val Glu Val Asn Ser Asn
115 120 125
Asp Ile Phe Glu Val Leu Lys Arg Ala Phe Gly Val Leu Lys Met Tyr
130 135 140
Arg Asp Leu Thr Asn Ala Tyr Lys Thr Tyr Glu Glu Lys Leu Asn Asp
145 150 155 160
Gly Cys Glu Phe Leu Thr Ser Thr Glu Gln Pro Leu Ser Gly Met Ile
165 170 175
Asn Asn Tyr Tyr Thr Val Ala Leu Arg Asn Met Asn Glu Arg Tyr Gly
180 185 190
Tyr Lys Thr Glu Asp Leu Ala Phe Ile Gln Asp Lys Arg Phe Lys Phe
195 200 205
Val Lys Asp Ala Tyr Gly Lys Lys Lys Ser Gln Val Asn Thr Gly Phe
210 215 220
Phe Leu Ser Leu Gln Asp Tyr Asn Gly Asp Thr Gln Lys Lys Leu His
225 230 235 240
Leu Ser Gly Val Gly Ile Ala Leu Leu Ile Cys Leu Phe Leu Asp Lys
245 250 255
Gln Tyr Ile Asn Ile Phe Leu Ser Arg Leu Pro Ile Phe Ser Ser Tyr
260 265 270
Asn Ala Gln Ser Glu Glu Arg Arg Ile Ile Ile Arg Ser Phe Gly Ile
275 280 285
Asn Ser Ile Lys Leu Pro Lys Asp Arg Ile His Ser Glu Lys Ser Asn
290 295 300
Lys Ser Val Ala Met Asp Met Leu Asn Glu Val Lys Arg Cys Pro Asp
305 310 315 320
Glu Leu Phe Thr Thr Leu Ser Ala Glu Lys Gln Ser Arg Phe Arg Ile
325 330 335
Ile Ser Asp Asp His Asn Glu Val Leu Met Lys Arg Ser Ser Asp Arg
340 345 350
Phe Val Pro Leu Leu Leu Gln Tyr Ile Asp Tyr Gly Lys Leu Phe Asp
355 360 365
His Ile Arg Phe His Val Asn Met Gly Lys Leu Arg Tyr Leu Leu Lys
370 375 380
Ala Asp Lys Thr Cys Ile Asp Gly Gln Thr Arg Val Arg Val Ile Glu
385 390 395 400
Gln Pro Leu Asn Gly Phe Gly Arg Leu Glu Glu Ala Glu Thr Met Arg
405 410 415
Lys Gln Glu Asn Gly Thr Phe Gly Asn Ser Gly Ile Arg Ile Arg Asp
420 425 430
Phe Glu Asn Met Lys Arg Asp Asp Ala Asn Pro Ala Asn Tyr Pro Tyr
435 440 445
Ile Val Asp Thr Tyr Thr His Tyr Ile Leu Glu Asn Asn Lys Val Glu
450 455 460
Met Phe Ile Asn Asp Lys Glu Asp Ser Ala Pro Leu Leu Pro Val Ile
465 470 475 480
Glu Asp Asp Arg Tyr Val Val Lys Thr Ile Pro Ser Cys Arg Met Ser
485 490 495
Thr Leu Glu Ile Pro Ala Met Ala Phe His Met Phe Leu Phe Gly Ser
500 505 510
Lys Lys Thr Glu Lys Leu Ile Val Asp Val His Asn Arg Tyr Lys Arg
515 520 525
Leu Phe Gln Ala Met Gln Lys Glu Glu Val Thr Ala Glu Asn Ile Ala
530 535 540
Ser Phe Gly Ile Ala Glu Ser Asp Leu Pro Gln Lys Ile Leu Asp Leu
545 550 555 560
Ile Ser Gly Asn Ala His Gly Lys Asp Val Asp Ala Phe Ile Arg Leu
565 570 575
Thr Val Asp Asp Met Leu Thr Asp Thr Glu Arg Arg Ile Lys Arg Phe
580 585 590
Lys Asp Asp Arg Lys Ser Ile Arg Ser Ala Asp Asn Lys Met Gly Lys
595 600 605
Arg Gly Phe Lys Gln Ile Ser Thr Gly Lys Leu Ala Asp Phe Leu Ala
610 615 620
Lys Asp Ile Val Leu Phe Gln Pro Ser Val Asn Asp Gly Glu Asn Lys
625 630 635 640
Ile Thr Gly Leu Asn Tyr Arg Ile Met Gln Ser Ala Ile Ala Val Tyr
645 650 655
Asp Ser Gly Asp Asp Tyr Glu Ala Lys Gln Gln Phe Lys Leu Met Phe
660 665 670
Glu Lys Ala Arg Leu Ile Gly Lys Gly Thr Thr Glu Pro His Pro Phe
675 680 685
Leu Tyr Lys Val Phe Ala Arg Ser Ile Pro Ala Asn Ala Val Glu Phe
690 695 700
Tyr Glu Arg Tyr Leu Ile Glu Arg Lys Phe Tyr Leu Thr Gly Leu Ser
705 710 715 720
Asn Glu Ile Lys Lys Gly Asn Arg Val Asp Val Pro Phe Ile Arg Arg
725 730 735
Asp Gln Asn Lys Trp Lys Thr Pro Ala Met Lys Thr Leu Gly Arg Ile
740 745 750
Tyr Ser Glu Asp Leu Pro Val Glu Leu Pro Arg Gln Met Phe Asp Asn
755 760 765
Glu Ile Lys Ser His Leu Lys Ser Leu Pro Gln Met Glu Gly Ile Asp
770 775 780
Phe Asn Asn Ala Asn Val Thr Tyr Leu Ile Ala Glu Tyr Met Lys Arg
785 790 795 800
Val Leu Asp Asp Asp Phe Gln Thr Phe Tyr Gln Trp Asn Arg Asn Tyr
805 810 815
Arg Tyr Met Asp Met Leu Lys Gly Glu Tyr Asp Arg Lys Gly Ser Leu
820 825 830
Gln His Cys Phe Thr Ser Val Glu Glu Arg Glu Gly Leu Trp Lys Glu
835 840 845
Arg Ala Ser Arg Thr Glu Arg Tyr Arg Lys Gln Ala Ser Asn Lys Ile
850 855 860
Arg Ser Asn Arg Gln Met Arg Asn Ala Ser Ser Glu Glu Ile Glu Thr
865 870 875 880
Ile Leu Asp Lys Arg Leu Ser Asn Ser Arg Asn Glu Tyr Gln Lys Ser
885 890 895
Glu Lys Val Ile Arg Arg Tyr Arg Val Gln Asp Ala Leu Leu Phe Leu
900 905 910
Leu Ala Lys Lys Thr Leu Thr Glu Leu Ala Asp Phe Asp Gly Glu Arg
915 920 925
Phe Lys Leu Lys Glu Ile Met Pro Asp Ala Glu Lys Gly Ile Leu Ser
930 935 940
Glu Ile Met Pro Met Ser Phe Thr Phe Glu Lys Gly Gly Lys Lys Tyr
945 950 955 960
Thr Ile Thr Ser Glu Gly Met Lys Leu Lys Asn Tyr Gly Asp Phe Phe
965 970 975
Val Leu Ala Ser Asp Lys Arg Ile Gly Asn Leu Leu Glu Leu Val Gly
980 985 990
Ser Asp Ile Val Ser Lys Glu Asp Gly Ser Leu Gln Leu Pro Pro Leu
995 1000 1005
Glu Arg Leu Thr Leu Gly Ser Gly Ser Ser Met Glu Ala Ser Pro
1010 1015 1020
Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser
1025 1030 1035
Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr
1040 1045 1050
Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln
1055 1060 1065
His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly
1070 1075 1080
Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro
1085 1090 1095
Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe
1100 1105 1110
Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val
1115 1120 1125
Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe
1130 1135 1140
Ala Ala Arg Ile Tyr Asp Asp Asp Pro Leu Tyr Lys Glu Ala Leu
1145 1150 1155
Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr
1160 1165 1170
Asp Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly
1175 1180 1185
Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala
1190 1195 1200
Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn
1205 1210 1215
<210> 9
<211> 1225
<212> PRT
<213> Artificial Sequence
<220>
<223> CURE-X
<400> 9
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Gly Ser Ile Glu Lys Lys Lys Ser Phe Ala Lys Gly Met
20 25 30
Gly Val Lys Ser Thr Leu Val Ser Gly Ser Lys Val Tyr Met Thr Thr
35 40 45
Phe Ala Glu Gly Ser Asp Ala Arg Leu Glu Lys Ile Val Glu Gly Asp
50 55 60
Ser Ile Arg Ser Val Asn Glu Gly Glu Ala Phe Ser Ala Glu Met Ala
65 70 75 80
Asp Lys Asn Ala Gly Tyr Lys Ile Gly Asn Ala Lys Phe Ser His Pro
85 90 95
Lys Gly Tyr Ala Val Val Ala Asn Asn Pro Leu Tyr Thr Gly Pro Val
100 105 110
Gln Gln Asp Met Leu Gly Leu Lys Glu Thr Leu Glu Lys Arg Tyr Phe
115 120 125
Gly Glu Ser Ala Asp Gly Asn Asp Asn Ile Cys Ile Gln Val Ile His
130 135 140
Asn Ile Leu Asp Ile Glu Lys Ile Leu Ala Glu Tyr Ile Thr Asn Ala
145 150 155 160
Ala Tyr Ala Val Asn Asn Ile Ser Gly Leu Asp Lys Asp Ile Ile Gly
165 170 175
Phe Gly Lys Phe Ser Thr Val Tyr Thr Tyr Asp Glu Phe Lys Asp Pro
180 185 190
Glu His His Arg Ala Ala Phe Asn Asn Asn Asp Lys Leu Ile Asn Ala
195 200 205
Ile Lys Ala Gln Tyr Asp Glu Phe Asp Asn Phe Leu Asp Asn Pro Arg
210 215 220
Leu Gly Tyr Phe Gly Gln Ala Phe Phe Ser Lys Glu Gly Arg Asn Tyr
225 230 235 240
Ile Ile Asn Tyr Gly Asn Glu Cys Tyr Asp Ile Leu Ala Leu Leu Ser
245 250 255
Gly Leu Ala His Trp Val Val Ala Asn Asn Glu Glu Glu Ser Arg Ile
260 265 270
Ser Arg Thr Trp Leu Tyr Asn Leu Asp Lys Asn Leu Asp Asn Glu Tyr
275 280 285
Ile Ser Thr Leu Asn Tyr Leu Tyr Asp Arg Ile Thr Asn Glu Leu Thr
290 295 300
Asn Ser Phe Ser Lys Asn Ser Ala Ala Asn Val Asn Tyr Ile Ala Glu
305 310 315 320
Thr Leu Gly Ile Asn Pro Ala Glu Phe Ala Glu Gln Tyr Phe Arg Phe
325 330 335
Ser Ile Met Lys Glu Gln Lys Asn Leu Gly Phe Asn Ile Thr Lys Leu
340 345 350
Arg Glu Val Met Leu Asp Arg Lys Asp Met Ser Glu Ile Arg Lys Asn
355 360 365
His Lys Val Phe Asp Ser Ile Arg Thr Lys Val Tyr Thr Met Met Asp
370 375 380
Phe Val Ile Tyr Arg Tyr Tyr Ile Glu Glu Asp Ala Lys Val Ala Ala
385 390 395 400
Ala Asn Lys Ser Leu Pro Asp Asn Glu Lys Ser Leu Ser Glu Lys Asp
405 410 415
Ile Phe Val Ile Asn Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys Asp
420 425 430
Ala Leu Tyr Tyr Asp Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu Asn
435 440 445
Ile Met His Asn Ile Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu Tyr
450 455 460
Lys Lys Lys Asp Ala Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly Arg
465 470 475 480
Asp Val Ser Ala Phe Ser Lys Leu Met Tyr Ala Leu Thr Met Phe Leu
485 490 495
Asp Gly Lys Glu Ile Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe
500 505 510
Asp Asn Ile Gln Ser Phe Leu Lys Val Met Pro Leu Ile Gly Val Asn
515 520 525
Ala Lys Phe Val Glu Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys Ile
530 535 540
Ala Asp Glu Leu Arg Leu Ile Lys Ser Phe Ala Arg Met Gly Glu Pro
545 550 555 560
Ile Ala Asp Ala Arg Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile Leu
565 570 575
Gly Thr Asn Gly Thr Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu
580 585 590
Ser Pro Lys Lys Lys Arg Lys Val Gly Pro Gly Ser Met Glu Ala Ser
595 600 605
Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser
610 615 620
Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr Glu
625 630 635 640
Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln His Arg
645 650 655
Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly
660 665 670
Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu Gln Leu
675 680 685
Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro
690 695 700
Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe Leu Gln Glu
705 710 715 720
Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg Ile Tyr Asp Asp
725 730 735
Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp Ala Gly Ala
740 745 750
Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His Cys Trp Asp Thr
755 760 765
Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp
770 775 780
Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn
785 790 795 800
Gln Gly Asn Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser
805 810 815
Pro Lys Lys Lys Arg Lys Val Gly Ser Met Arg Asn Phe Ile Ile Asn
820 825 830
Asn Val Ile Ser Asn Lys Arg Phe His Tyr Leu Ile Arg Tyr Gly Asp
835 840 845
Pro Ala His Leu His Glu Ile Ala Lys Asn Glu Ala Val Val Lys Phe
850 855 860
Val Leu Gly Arg Ile Ala Asp Ile Gln Lys Lys Gln Gly Gln Asn Gly
865 870 875 880
Lys Asn Gln Ile Asp Arg Tyr Tyr Glu Thr Cys Ile Gly Lys Asp Lys
885 890 895
Gly Lys Ser Val Ser Glu Lys Val Asp Ala Leu Thr Lys Ile Ile Thr
900 905 910
Gly Met Asn Tyr Asp Gln Phe Asp Lys Lys Arg Ser Val Ile Glu Asp
915 920 925
Thr Gly Arg Glu Asn Ala Glu Arg Glu Lys Phe Lys Lys Ile Ile Ser
930 935 940
Leu Tyr Leu Thr Val Ile Tyr His Ile Leu Lys Asn Ile Val Asn Ile
945 950 955 960
Asn Ala Arg Tyr Val Ile Gly Phe His Cys Val Glu Arg Asp Ala Gln
965 970 975
Leu Tyr Lys Glu Lys Gly Tyr Asp Ile Asn Leu Lys Lys Leu Glu Glu
980 985 990
Lys Gly Phe Ser Ser Val Thr Lys Leu Cys Ala Gly Ile Asp Glu Thr
995 1000 1005
Ala Pro Asp Lys Arg Lys Asp Val Glu Lys Glu Met Ala Glu Arg
1010 1015 1020
Ala Lys Glu Ser Ile Asp Ser Leu Glu Ser Ala Asn Pro Lys Leu
1025 1030 1035
Tyr Ala Asn Tyr Ile Lys Tyr Ser Asp Glu Lys Lys Ala Glu Glu
1040 1045 1050
Phe Thr Arg Gln Ile Asn Arg Glu Lys Ala Lys Thr Ala Leu Asn
1055 1060 1065
Ala Tyr Leu Arg Asn Thr Lys Trp Asn Val Ile Ile Arg Glu Asp
1070 1075 1080
Leu Leu Arg Ile Asp Asn Lys Thr Cys Thr Leu Phe Ala Asn Lys
1085 1090 1095
Ala Val Ala Leu Glu Val Ala Arg Tyr Val His Ala Tyr Ile Asn
1100 1105 1110
Asp Ile Ala Glu Val Asn Ser Tyr Phe Gln Leu Tyr His Tyr Ile
1115 1120 1125
Met Gln Arg Ile Ile Met Asn Glu Arg Tyr Glu Lys Ser Ser Gly
1130 1135 1140
Lys Val Ser Glu Tyr Phe Asp Ala Val Asn Asp Glu Lys Lys Tyr
1145 1150 1155
Asn Asp Arg Leu Leu Lys Leu Leu Cys Val Pro Phe Gly Tyr Cys
1160 1165 1170
Ile Pro Arg Phe Lys Asn Leu Ser Ile Glu Ala Leu Phe Asp Arg
1175 1180 1185
Asn Glu Ala Ala Lys Phe Asp Lys Glu Lys Lys Lys Val Ser Gly
1190 1195 1200
Asn Ser Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser
1205 1210 1215
Pro Lys Lys Lys Arg Lys Val
1220 1225
<210> 10
<211> 1239
<212> PRT
<213> Artificial Sequence
<220>
<223> CURE-N
<400> 10
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val Asn Ile Pro Ala Leu Val Glu Asn Gln Lys Lys Tyr Phe Gly
20 25 30
Thr Tyr Ser Val Met Ala Met Leu Asn Ala Gln Thr Val Leu Asp His
35 40 45
Ile Gln Lys Val Ala Asp Ile Glu Gly Glu Gln Asn Glu Asn Asn Glu
50 55 60
Asn Leu Trp Phe His Pro Val Met Ser His Leu Tyr Asn Ala Lys Asn
65 70 75 80
Gly Tyr Asp Lys Gln Pro Glu Lys Thr Met Phe Ile Ile Glu Arg Leu
85 90 95
Gln Ser Tyr Phe Pro Phe Leu Lys Ile Met Ala Glu Asn Gln Arg Glu
100 105 110
Tyr Ser Asn Gly Lys Tyr Lys Gln Asn Arg Val Glu Val Asn Ser Asn
115 120 125
Asp Ile Phe Glu Val Leu Lys Arg Ala Phe Gly Val Leu Lys Met Tyr
130 135 140
Arg Asp Leu Thr Asn Ala Tyr Lys Thr Tyr Glu Glu Lys Leu Asn Asp
145 150 155 160
Gly Cys Glu Phe Leu Thr Ser Thr Glu Gln Pro Leu Ser Gly Met Ile
165 170 175
Asn Asn Tyr Tyr Thr Val Ala Leu Arg Asn Met Asn Glu Arg Tyr Gly
180 185 190
Tyr Lys Thr Glu Asp Leu Ala Phe Ile Gln Asp Lys Arg Phe Lys Phe
195 200 205
Val Lys Asp Ala Tyr Gly Lys Lys Lys Ser Gln Val Asn Thr Gly Phe
210 215 220
Phe Leu Ser Leu Gln Asp Tyr Asn Gly Asp Thr Gln Lys Lys Leu His
225 230 235 240
Leu Ser Gly Val Gly Ile Ala Leu Leu Ile Cys Leu Phe Leu Asp Lys
245 250 255
Gln Tyr Ile Asn Ile Phe Leu Ser Arg Leu Pro Ile Phe Ser Ser Tyr
260 265 270
Asn Ala Gln Ser Glu Glu Arg Arg Ile Ile Ile Arg Ser Phe Gly Ile
275 280 285
Asn Ser Ile Lys Leu Pro Lys Asp Arg Ile His Ser Glu Lys Ser Asn
290 295 300
Lys Ser Val Ala Met Asp Met Leu Asn Glu Val Lys Arg Cys Pro Asp
305 310 315 320
Glu Leu Phe Thr Thr Leu Ser Ala Glu Lys Gln Ser Arg Phe Arg Ile
325 330 335
Ile Ser Asp Asp His Asn Glu Val Leu Met Lys Arg Ser Ser Asp Arg
340 345 350
Phe Val Pro Leu Leu Leu Gln Tyr Ile Asp Tyr Gly Lys Leu Phe Asp
355 360 365
His Ile Arg Phe His Val Asn Met Gly Lys Leu Arg Tyr Leu Leu Lys
370 375 380
Ala Asp Lys Thr Cys Ile Asp Gly Gln Thr Arg Val Arg Val Ile Glu
385 390 395 400
Gln Pro Leu Asn Gly Phe Gly Arg Leu Glu Glu Ala Glu Thr Met Arg
405 410 415
Lys Gln Glu Asn Gly Thr Phe Gly Asn Ser Gly Ile Arg Ile Arg Asp
420 425 430
Phe Glu Asn Met Lys Arg Asp Asp Ala Asn Pro Ala Asn Tyr Pro Tyr
435 440 445
Ile Val Asp Thr Tyr Thr His Tyr Ile Leu Glu Asn Asn Lys Val Glu
450 455 460
Met Phe Ile Asn Asp Lys Glu Asp Ser Ala Pro Leu Leu Pro Val Ile
465 470 475 480
Glu Asp Asp Arg Tyr Val Val Lys Thr Ile Pro Ser Cys Arg Met Ser
485 490 495
Thr Leu Glu Ile Pro Ala Met Ala Phe His Met Phe Leu Phe Gly Ser
500 505 510
Lys Lys Thr Glu Lys Leu Ile Val Asp Val His Asn Arg Tyr Lys Arg
515 520 525
Leu Phe Gln Ala Met Gln Lys Glu Glu Val Thr Ala Glu Asn Ile Ala
530 535 540
Ser Phe Gly Ile Ala Glu Ser Asp Leu Pro Gln Lys Ile Leu Asp Leu
545 550 555 560
Ile Ser Gly Asn Ala His Gly Lys Asp Val Asp Ala Phe Ile Arg Leu
565 570 575
Thr Val Asp Asp Met Leu Thr Asp Thr Glu Arg Arg Ile Lys Arg Phe
580 585 590
Lys Asp Asp Arg Lys Ser Ile Arg Ser Ala Asp Asn Lys Met Gly Lys
595 600 605
Arg Gly Phe Lys Gln Ile Ser Thr Gly Lys Leu Ala Asp Phe Leu Ala
610 615 620
Lys Asp Ile Val Leu Phe Gln Pro Ser Val Asn Asp Gly Glu Asn Lys
625 630 635 640
Ile Thr Gly Leu Asn Tyr Arg Ile Met Gln Ser Ala Ile Ala Val Tyr
645 650 655
Asp Ser Gly Asp Asp Tyr Glu Ala Lys Gln Gln Phe Lys Leu Met Phe
660 665 670
Glu Lys Ala Arg Leu Ile Gly Lys Gly Thr Thr Glu Pro His Pro Phe
675 680 685
Leu Tyr Lys Val Phe Ala Arg Ser Ile Pro Ala Asn Ala Val Glu Phe
690 695 700
Tyr Glu Arg Tyr Leu Ile Glu Arg Lys Phe Tyr Leu Thr Gly Leu Ser
705 710 715 720
Asn Glu Ile Lys Lys Gly Asn Arg Val Asp Val Pro Phe Ile Arg Arg
725 730 735
Asp Gln Asn Lys Trp Lys Thr Pro Ala Met Lys Thr Leu Gly Arg Ile
740 745 750
Tyr Ser Glu Asp Leu Pro Val Glu Leu Pro Arg Gln Met Phe Asp Asn
755 760 765
Glu Ile Lys Ser His Leu Lys Ser Leu Pro Gln Met Glu Gly Ile Asp
770 775 780
Phe Asn Asn Ala Asn Val Thr Tyr Leu Ile Ala Glu Tyr Met Lys Arg
785 790 795 800
Val Leu Asp Asp Asp Phe Gln Thr Phe Tyr Gln Trp Asn Arg Asn Tyr
805 810 815
Arg Tyr Met Asp Met Leu Lys Gly Glu Tyr Asp Arg Lys Gly Ser Leu
820 825 830
Gln His Cys Phe Thr Ser Val Glu Glu Arg Glu Gly Leu Trp Lys Glu
835 840 845
Arg Ala Ser Arg Thr Glu Arg Tyr Arg Lys Gln Ala Ser Asn Lys Ile
850 855 860
Arg Ser Asn Arg Gln Met Arg Asn Ala Ser Ser Glu Glu Ile Glu Thr
865 870 875 880
Ile Leu Asp Lys Arg Leu Ser Asn Ser Arg Asn Glu Tyr Gln Lys Ser
885 890 895
Glu Lys Val Ile Arg Arg Tyr Arg Val Gln Asp Ala Leu Leu Phe Leu
900 905 910
Leu Ala Lys Lys Thr Leu Thr Glu Leu Ala Asp Phe Asp Gly Glu Arg
915 920 925
Phe Lys Leu Lys Glu Ile Met Pro Asp Ala Glu Lys Gly Ile Leu Ser
930 935 940
Glu Ile Met Pro Met Ser Phe Thr Phe Glu Lys Gly Gly Lys Lys Tyr
945 950 955 960
Thr Ile Thr Ser Glu Gly Met Lys Leu Lys Asn Tyr Gly Asp Phe Phe
965 970 975
Val Leu Ala Ser Asp Lys Arg Ile Gly Asn Leu Leu Glu Leu Val Gly
980 985 990
Ser Asp Ile Val Ser Lys Glu Asp Gly Ser Leu Gln Leu Pro Pro Leu
995 1000 1005
Glu Arg Leu Thr Leu Gly Ser Gly Ser Ser Met Glu Ala Ser Pro
1010 1015 1020
Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser
1025 1030 1035
Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr
1040 1045 1050
Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln
1055 1060 1065
His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly
1070 1075 1080
Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro
1085 1090 1095
Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe
1100 1105 1110
Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val
1115 1120 1125
Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe
1130 1135 1140
Ala Ala Arg Ile Tyr Asp Asp Asp Pro Leu Tyr Lys Glu Ala Leu
1145 1150 1155
Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr
1160 1165 1170
Asp Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly
1175 1180 1185
Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala
1190 1195 1200
Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn Ala
1205 1210 1215
Ala Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro
1220 1225 1230
Lys Lys Lys Arg Lys Val
1235
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> 4276F
<400> 11
ggcgtaatca tggtcatagc tg 22
<210> 12
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 4276R
<400> 12
gctcgagccg gatcccagtg tcagtctttc aa 32
<210> 13
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 4191F
<400> 13
ggatccggct cgagcatgga agccagccca gca 33
<210> 14
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> 4191R
<400> 14
atccagcggc cgcgtttccc tgattctgga gaatg 35
<210> 15
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 4275F
<400> 15
acgcggccgc tggatccgct actaacttca gcc 33
<210> 16
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> 4275R
<400> 16
gaccatgatt acgccttcga ggtcgacggt atcgatg 37
<210> 17
<211> 8139
<212> DNA
<213> Artificial Sequence
<220>
<223> TC979: CMV dPspCas13b-Apobec3A序列
<400> 17
ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 60
caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 120
cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 180
gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 240
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 300
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 360
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 420
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 480
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 540
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 600
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 660
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 720
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 780
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 840
cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 900
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtttttttgt 960
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 1020
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 1080
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 1140
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 1200
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 1260
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 1320
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 1380
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 1440
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 1500
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 1560
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 1620
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 1680
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 1740
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 1800
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 1860
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 1920
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 1980
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 2040
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 2100
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 2160
tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc 2220
tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag 2280
tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag 2340
aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt 2400
tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 2460
ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 2520
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 2580
actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 2640
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 2700
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 2760
ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 2820
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 2880
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 2940
atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga 3000
gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct 3060
ggctagcgtt taaacttaag cttgccacca tgaacatccc cgctctggtg gaaaaccaga 3120
agaagtactt tggcacctac agcgtgatgg ccatgctgaa cgctcagacc gtgctggacc 3180
acatccagaa ggtggccgat attgagggcg agcagaacga gaacaacgag aatctgtggt 3240
ttcaccccgt gatgagccac ctgtacaacg ccaagaacgg ctacgacaag cagcccgaga 3300
aaaccatgtt catcatcgag cggctgcaga gctacttccc attcctgaag atcatggccg 3360
agaaccagag agagtacagc aacggcaagt acaagcagaa ccgcgtggaa gtgaacagca 3420
acgacatctt cgaggtgctg aagcgcgcct tcggcgtgct gaagatgtac agggacctga 3480
ccaacgcata caagacctac gaggaaaagc tgaacgacgg ctgcgagttc ctgaccagca 3540
cagagcaacc tctgagcggc atgatcaaca actactacac agtggccctg cggaacatga 3600
acgagagata cggctacaag acagaggacc tggccttcat ccaggacaag cggttcaagt 3660
tcgtgaagga cgcctacggc aagaaaaagt cccaagtgaa taccggattc ttcctgagcc 3720
tgcaggacta caacggcgac acacagaaga agctgcacct gagcggagtg ggaatcgccc 3780
tgctgatctg cctgttcctg gacaagcagt acatcaacat ctttctgagc aggctgccca 3840
tcttctccag ctacaatgcc cagagcgagg aacggcggat catcatcaga tccttcggca 3900
tcaacagcat caagctgccc aaggaccgga tccacagcga gaagtccaac aagagcgtgg 3960
ccatggatat gctcaacgaa gtgaagcggt gccccgacga gctgttcaca acactgtctg 4020
ccgagaagca gtcccggttc agaatcatca gcgacgacca caatgaagtg ctgatgaagc 4080
ggagcagcga cagattcgtg cctctgctgc tgcagtatat cgattacggc aagctgttcg 4140
accacatcag gttccacgtg aacatgggca agctgagata cctgctgaag gccgacaaga 4200
cctgcatcga cggccagacc agagtcagag tgatcgagca gcccctgaac ggcttcggca 4260
gactggaaga ggccgagaca atgcggaagc aagagaacgg caccttcggc aacagcggca 4320
tccggatcag agacttcgag aacatgaagc gggacgacgc caatcctgcc aactatccct 4380
acatcgtgga cacctacaca cactacatcc tggaaaacaa caaggtcgag atgtttatca 4440
acgacaaaga ggacagcgcc ccactgctgc ccgtgatcga ggatgataga tacgtggtca 4500
agacaatccc cagctgccgg atgagcaccc tggaaattcc agccatggcc ttccacatgt 4560
ttctgttcgg cagcaagaaa accgagaagc tgatcgtgga cgtgcacaac cggtacaaga 4620
gactgttcca ggccatgcag aaagaagaag tgaccgccga gaatatcgcc agcttcggaa 4680
tcgccgagag cgacctgcct cagaagatcc tggatctgat cagcggcaat gcccacggca 4740
aggatgtgga cgccttcatc agactgaccg tggacgacat gctgaccgac accgagcgga 4800
gaatcaagag attcaaggac gaccggaagt ccattcggag cgccgacaac aagatgggaa 4860
agagaggctt caagcagatc tccacaggca agctggccga cttcctggcc aaggacatcg 4920
tgctgtttca gcccagcgtg aacgatggcg agaacaagat caccggcctg aactaccgga 4980
tcatgcagag cgccattgcc gtgtacgata gcggcgacga ttacgaggcc aagcagcagt 5040
tcaagctgat gttcgagaag gcccggctga tcggcaaggg cacaacagag cctcatccat 5100
ttctgtacaa ggtgttcgcc cgcagcatcc ccgccaatgc cgtcgagttc tacgagcgct 5160
acctgatcga gcggaagttc tacctgaccg gcctgtccaa cgagatcaag aaaggcaaca 5220
gagtggatgt gcccttcatc cggcgggacc agaacaagtg gaaaacaccc gccatgaaga 5280
ccctgggcag aatctacagc gaggatctgc ccgtggaact gcccagacag atgttcgaca 5340
atgagatcaa gtcccacctg aagtccctgc cacagatgga aggcatcgac ttcaacaatg 5400
ccaacgtgac ctatctgatc gccgagtaca tgaagagagt gctggacgac gacttccaga 5460
ccttctacca gtggaaccgc aactaccggt acatggacat gcttaagggc gagtacgaca 5520
gaaagggctc cctgcagcac tgcttcacca gcgtggaaga gagagaaggc ctctggaaag 5580
agcgggcctc cagaacagag cggtacagaa agcaggccag caacaagatc cgcagcaacc 5640
ggcagatgag aaacgccagc agcgaagaga tcgagacaat cctggataag cggctgagca 5700
acagccggaa cgagtaccag aaaagcgaga aagtgatccg gcgctacaga gtgcaggatg 5760
ccctgctgtt tctgctggcc aaaaagaccc tgaccgaact ggccgatttc gacggcgaga 5820
ggttcaaact gaaagaaatc atgcccgacg ccgagaaggg aatcctgagc gagatcatgc 5880
ccatgagctt caccttcgag aaaggcggca agaagtacac catcaccagc gagggcatga 5940
agctgaagaa ctacggcgac ttctttgtgc tggctagcga caagaggatc ggcaacctgc 6000
tggaactcgt gggcagcgac atcgtgtcca aagaggatgg atcccttcaa ctgcctccac 6060
ttgaaagact gacactggga tccggctcga gcatggaagc cagcccagca tccgggccca 6120
gacacttgat ggatccacac atattcactt ccaactttaa caatggcatt ggaaggcata 6180
agacctacct gtgctacgaa gtggagcgcc tggacaatgg cacctcggtc aagatggacc 6240
agcacagggg ctttctacac aaccaggcta agaatcttct ctgtggcttt tacggccgcc 6300
atgcggagct gcgcttcttg gacctggttc cttctttgca gttggacccg gcccagatct 6360
acagggtcac ttggttcatc tcctggagcc cctgcttctc ctggggctgt gccggggaag 6420
tgcgtgcgtt ccttcaggag aacacacacg tgagactgcg tatcttcgct gcccgcatct 6480
atgattacga ccccctatat aaggaggcac tgcaaatgct gcgggatgct ggggcccaag 6540
tctccatcat gacctacgat gaatttaagc actgctggga cacctttgtg gaccaccagg 6600
gatgtccctt ccagccctgg gatggactag atgagcacag ccaagccctg agtgggaggc 6660
tgcgggccat tctccagaat cagggaaacg cggccgctgg atccgctact aacttcagcc 6720
tgctgaagca ggctggagac gtggaggaga accctggacc tatggtgagc aagggcgagg 6780
aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg 6840
tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc 6900
agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt 6960
cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact 7020
acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg 7080
gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg 7140
tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg 7200
gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca 7260
agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca 7320
aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca 7380
cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact 7440
ccaccggcgg catggacgag ctgtacaagt aaggccgcga ctctagagtc gacctgcagg 7500
catgcaagct tgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa 7560
gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 7620
tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 7680
cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 7740
gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc 7800
tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 7860
ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 7920
ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga 7980
cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 8040
tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc 8100
tttgggccgc ctccccgcat cgataccgtc gacctcgaa 8139
<210> 18
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> 4676F
<400> 18
gcatgcaagc ttgataatca acctctggat tacaaaattt g 41
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 4676R
<400> 19
agtgagcgag gaagctcccc agcatgcctg 30
<210> 20
<211> 8093
<212> DNA
<213> Artificial Sequence
<220>
<223> TC1116: CMV dPspCas13b-Apobec3A序列
<400> 20
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttgcca ccatgaacat ccccgctctg gtggaaaacc agaagaagta 960
ctttggcacc tacagcgtga tggccatgct gaacgctcag accgtgctgg accacatcca 1020
gaaggtggcc gatattgagg gcgagcagaa cgagaacaac gagaatctgt ggtttcaccc 1080
cgtgatgagc cacctgtaca acgccaagaa cggctacgac aagcagcccg agaaaaccat 1140
gttcatcatc gagcggctgc agagctactt cccattcctg aagatcatgg ccgagaacca 1200
gagagagtac agcaacggca agtacaagca gaaccgcgtg gaagtgaaca gcaacgacat 1260
cttcgaggtg ctgaagcgcg ccttcggcgt gctgaagatg tacagggacc tgaccaacgc 1320
atacaagacc tacgaggaaa agctgaacga cggctgcgag ttcctgacca gcacagagca 1380
acctctgagc ggcatgatca acaactacta cacagtggcc ctgcggaaca tgaacgagag 1440
atacggctac aagacagagg acctggcctt catccaggac aagcggttca agttcgtgaa 1500
ggacgcctac ggcaagaaaa agtcccaagt gaataccgga ttcttcctga gcctgcagga 1560
ctacaacggc gacacacaga agaagctgca cctgagcgga gtgggaatcg ccctgctgat 1620
ctgcctgttc ctggacaagc agtacatcaa catctttctg agcaggctgc ccatcttctc 1680
cagctacaat gcccagagcg aggaacggcg gatcatcatc agatccttcg gcatcaacag 1740
catcaagctg cccaaggacc ggatccacag cgagaagtcc aacaagagcg tggccatgga 1800
tatgctcaac gaagtgaagc ggtgccccga cgagctgttc acaacactgt ctgccgagaa 1860
gcagtcccgg ttcagaatca tcagcgacga ccacaatgaa gtgctgatga agcggagcag 1920
cgacagattc gtgcctctgc tgctgcagta tatcgattac ggcaagctgt tcgaccacat 1980
caggttccac gtgaacatgg gcaagctgag atacctgctg aaggccgaca agacctgcat 2040
cgacggccag accagagtca gagtgatcga gcagcccctg aacggcttcg gcagactgga 2100
agaggccgag acaatgcgga agcaagagaa cggcaccttc ggcaacagcg gcatccggat 2160
cagagacttc gagaacatga agcgggacga cgccaatcct gccaactatc cctacatcgt 2220
ggacacctac acacactaca tcctggaaaa caacaaggtc gagatgttta tcaacgacaa 2280
agaggacagc gccccactgc tgcccgtgat cgaggatgat agatacgtgg tcaagacaat 2340
ccccagctgc cggatgagca ccctggaaat tccagccatg gccttccaca tgtttctgtt 2400
cggcagcaag aaaaccgaga agctgatcgt ggacgtgcac aaccggtaca agagactgtt 2460
ccaggccatg cagaaagaag aagtgaccgc cgagaatatc gccagcttcg gaatcgccga 2520
gagcgacctg cctcagaaga tcctggatct gatcagcggc aatgcccacg gcaaggatgt 2580
ggacgccttc atcagactga ccgtggacga catgctgacc gacaccgagc ggagaatcaa 2640
gagattcaag gacgaccgga agtccattcg gagcgccgac aacaagatgg gaaagagagg 2700
cttcaagcag atctccacag gcaagctggc cgacttcctg gccaaggaca tcgtgctgtt 2760
tcagcccagc gtgaacgatg gcgagaacaa gatcaccggc ctgaactacc ggatcatgca 2820
gagcgccatt gccgtgtacg atagcggcga cgattacgag gccaagcagc agttcaagct 2880
gatgttcgag aaggcccggc tgatcggcaa gggcacaaca gagcctcatc catttctgta 2940
caaggtgttc gcccgcagca tccccgccaa tgccgtcgag ttctacgagc gctacctgat 3000
cgagcggaag ttctacctga ccggcctgtc caacgagatc aagaaaggca acagagtgga 3060
tgtgcccttc atccggcggg accagaacaa gtggaaaaca cccgccatga agaccctggg 3120
cagaatctac agcgaggatc tgcccgtgga actgcccaga cagatgttcg acaatgagat 3180
caagtcccac ctgaagtccc tgccacagat ggaaggcatc gacttcaaca atgccaacgt 3240
gacctatctg atcgccgagt acatgaagag agtgctggac gacgacttcc agaccttcta 3300
ccagtggaac cgcaactacc ggtacatgga catgcttaag ggcgagtacg acagaaaggg 3360
ctccctgcag cactgcttca ccagcgtgga agagagagaa ggcctctgga aagagcgggc 3420
ctccagaaca gagcggtaca gaaagcaggc cagcaacaag atccgcagca accggcagat 3480
gagaaacgcc agcagcgaag agatcgagac aatcctggat aagcggctga gcaacagccg 3540
gaacgagtac cagaaaagcg agaaagtgat ccggcgctac agagtgcagg atgccctgct 3600
gtttctgctg gccaaaaaga ccctgaccga actggccgat ttcgacggcg agaggttcaa 3660
actgaaagaa atcatgcccg acgccgagaa gggaatcctg agcgagatca tgcccatgag 3720
cttcaccttc gagaaaggcg gcaagaagta caccatcacc agcgagggca tgaagctgaa 3780
gaactacggc gacttctttg tgctggctag cgacaagagg atcggcaacc tgctggaact 3840
cgtgggcagc gacatcgtgt ccaaagagga tggatccctt caactgcctc cacttgaaag 3900
actgacactg ggatccggct cgagcatgga agccagccca gcatccgggc ccagacactt 3960
gatggatcca cacatattca cttccaactt taacaatggc attggaaggc ataagaccta 4020
cctgtgctac gaagtggagc gcctggacaa tggcacctcg gtcaagatgg accagcacag 4080
gggctttcta cacaaccagg ctaagaatct tctctgtggc ttttacggcc gccatgcgga 4140
gctgcgcttc ttggacctgg ttccttcttt gcagttggac ccggcccaga tctacagggt 4200
cacttggttc atctcctgga gcccctgctt ctcctggggc tgtgccgggg aagtgcgtgc 4260
gttccttcag gagaacacac acgtgagact gcgtatcttc gctgcccgca tctatgatta 4320
cgacccccta tataaggagg cactgcaaat gctgcgggat gctggggccc aagtctccat 4380
catgacctac gatgaattta agcactgctg ggacaccttt gtggaccacc agggatgtcc 4440
cttccagccc tgggatggac tagatgagca cagccaagcc ctgagtggga ggctgcgggc 4500
cattctccag aatcagggaa acgcggccgc tggatccgct actaacttca gcctgctgaa 4560
gcaggctgga gacgtggagg agaaccctgg acctatggtg agcaagggcg aggaggataa 4620
catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg 4680
ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc 4740
caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca 4800
gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa 4860
gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt 4920
ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct 4980
gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga 5040
ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag 5100
gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa 5160
gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca 5220
caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg 5280
cggcatggac gagctgtaca agtaaggccg cgactctaga gtcgacctgc aggcatgcaa 5340
gcttgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta 5400
tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc 5460
ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga 5520
ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac 5580
ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc 5640
cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc 5700
tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg 5760
gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc 5820
ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc 5880
gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg 5940
ataccgtcga cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 6000
gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 6060
attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 6120
agcaaggggg aggattggga agacaatagc aggcatgctg gggagcttcc tcgctcactg 6180
actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 6240
tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 6300
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 6360
ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 6420
aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 6480
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 6540
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 6600
aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 6660
cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 6720
ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 6780
gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 6840
gctcttgatc cggcaaacaa accaccgctg gtagcggttt ttttgtttgc aagcagcaga 6900
ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 6960
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 7020
tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 7080
aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 7140
tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 7200
gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 7260
atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 7320
tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 7380
ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 7440
ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 7500
tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 7560
ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 7620
ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 7680
tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 7740
gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 7800
taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 7860
cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 7920
agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 7980
gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 8040
ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc 8093
<210> 21
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> 4531F
<400> 21
cactgggatc cggctcga 18
<210> 22
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> 4543R
<400> 22
atcatagatg cgggcag 17
<210> 23
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> 4539F
<400> 23
tgcccgcatc tatgatgacg acccccta 28
<210> 24
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> 4544R
<400> 24
agcaggctga agttagtag 19
<210> 25
<211> 8093
<212> DNA
<213> Artificial Sequence
<220>
<223> TC1132N: CMV dPspCas13b-Apobec3A(Y132D)序列
<400> 25
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttgcca ccatgaacat ccccgctctg gtggaaaacc agaagaagta 960
ctttggcacc tacagcgtga tggccatgct gaacgctcag accgtgctgg accacatcca 1020
gaaggtggcc gatattgagg gcgagcagaa cgagaacaac gagaatctgt ggtttcaccc 1080
cgtgatgagc cacctgtaca acgccaagaa cggctacgac aagcagcccg agaaaaccat 1140
gttcatcatc gagcggctgc agagctactt cccattcctg aagatcatgg ccgagaacca 1200
gagagagtac agcaacggca agtacaagca gaaccgcgtg gaagtgaaca gcaacgacat 1260
cttcgaggtg ctgaagcgcg ccttcggcgt gctgaagatg tacagggacc tgaccaacgc 1320
atacaagacc tacgaggaaa agctgaacga cggctgcgag ttcctgacca gcacagagca 1380
acctctgagc ggcatgatca acaactacta cacagtggcc ctgcggaaca tgaacgagag 1440
atacggctac aagacagagg acctggcctt catccaggac aagcggttca agttcgtgaa 1500
ggacgcctac ggcaagaaaa agtcccaagt gaataccgga ttcttcctga gcctgcagga 1560
ctacaacggc gacacacaga agaagctgca cctgagcgga gtgggaatcg ccctgctgat 1620
ctgcctgttc ctggacaagc agtacatcaa catctttctg agcaggctgc ccatcttctc 1680
cagctacaat gcccagagcg aggaacggcg gatcatcatc agatccttcg gcatcaacag 1740
catcaagctg cccaaggacc ggatccacag cgagaagtcc aacaagagcg tggccatgga 1800
tatgctcaac gaagtgaagc ggtgccccga cgagctgttc acaacactgt ctgccgagaa 1860
gcagtcccgg ttcagaatca tcagcgacga ccacaatgaa gtgctgatga agcggagcag 1920
cgacagattc gtgcctctgc tgctgcagta tatcgattac ggcaagctgt tcgaccacat 1980
caggttccac gtgaacatgg gcaagctgag atacctgctg aaggccgaca agacctgcat 2040
cgacggccag accagagtca gagtgatcga gcagcccctg aacggcttcg gcagactgga 2100
agaggccgag acaatgcgga agcaagagaa cggcaccttc ggcaacagcg gcatccggat 2160
cagagacttc gagaacatga agcgggacga cgccaatcct gccaactatc cctacatcgt 2220
ggacacctac acacactaca tcctggaaaa caacaaggtc gagatgttta tcaacgacaa 2280
agaggacagc gccccactgc tgcccgtgat cgaggatgat agatacgtgg tcaagacaat 2340
ccccagctgc cggatgagca ccctggaaat tccagccatg gccttccaca tgtttctgtt 2400
cggcagcaag aaaaccgaga agctgatcgt ggacgtgcac aaccggtaca agagactgtt 2460
ccaggccatg cagaaagaag aagtgaccgc cgagaatatc gccagcttcg gaatcgccga 2520
gagcgacctg cctcagaaga tcctggatct gatcagcggc aatgcccacg gcaaggatgt 2580
ggacgccttc atcagactga ccgtggacga catgctgacc gacaccgagc ggagaatcaa 2640
gagattcaag gacgaccgga agtccattcg gagcgccgac aacaagatgg gaaagagagg 2700
cttcaagcag atctccacag gcaagctggc cgacttcctg gccaaggaca tcgtgctgtt 2760
tcagcccagc gtgaacgatg gcgagaacaa gatcaccggc ctgaactacc ggatcatgca 2820
gagcgccatt gccgtgtacg atagcggcga cgattacgag gccaagcagc agttcaagct 2880
gatgttcgag aaggcccggc tgatcggcaa gggcacaaca gagcctcatc catttctgta 2940
caaggtgttc gcccgcagca tccccgccaa tgccgtcgag ttctacgagc gctacctgat 3000
cgagcggaag ttctacctga ccggcctgtc caacgagatc aagaaaggca acagagtgga 3060
tgtgcccttc atccggcggg accagaacaa gtggaaaaca cccgccatga agaccctggg 3120
cagaatctac agcgaggatc tgcccgtgga actgcccaga cagatgttcg acaatgagat 3180
caagtcccac ctgaagtccc tgccacagat ggaaggcatc gacttcaaca atgccaacgt 3240
gacctatctg atcgccgagt acatgaagag agtgctggac gacgacttcc agaccttcta 3300
ccagtggaac cgcaactacc ggtacatgga catgcttaag ggcgagtacg acagaaaggg 3360
ctccctgcag cactgcttca ccagcgtgga agagagagaa ggcctctgga aagagcgggc 3420
ctccagaaca gagcggtaca gaaagcaggc cagcaacaag atccgcagca accggcagat 3480
gagaaacgcc agcagcgaag agatcgagac aatcctggat aagcggctga gcaacagccg 3540
gaacgagtac cagaaaagcg agaaagtgat ccggcgctac agagtgcagg atgccctgct 3600
gtttctgctg gccaaaaaga ccctgaccga actggccgat ttcgacggcg agaggttcaa 3660
actgaaagaa atcatgcccg acgccgagaa gggaatcctg agcgagatca tgcccatgag 3720
cttcaccttc gagaaaggcg gcaagaagta caccatcacc agcgagggca tgaagctgaa 3780
gaactacggc gacttctttg tgctggctag cgacaagagg atcggcaacc tgctggaact 3840
cgtgggcagc gacatcgtgt ccaaagagga tggatccctt caactgcctc cacttgaaag 3900
actgacactg ggatccggct cgagcatgga agccagccca gcatccgggc ccagacactt 3960
gatggatcca cacatattca cttccaactt taacaatggc attggaaggc ataagaccta 4020
cctgtgctac gaagtggagc gcctggacaa tggcacctcg gtcaagatgg accagcacag 4080
gggctttcta cacaaccagg ctaagaatct tctctgtggc ttttacggcc gccatgcgga 4140
gctgcgcttc ttggacctgg ttccttcttt gcagttggac ccggcccaga tctacagggt 4200
cacttggttc atctcctgga gcccctgctt ctcctggggc tgtgccgggg aagtgcgtgc 4260
gttccttcag gagaacacac acgtgagact gcgtatcttc gctgcccgca tctatgatga 4320
cgacccccta tataaggagg cactgcaaat gctgcgggat gctggggccc aagtctccat 4380
catgacctac gatgaattta agcactgctg ggacaccttt gtggaccacc agggatgtcc 4440
cttccagccc tgggatggac tagatgagca cagccaagcc ctgagtggga ggctgcgggc 4500
cattctccag aatcagggaa acgcggccgc tggatccgct actaacttca gcctgctgaa 4560
gcaggctgga gacgtggagg agaaccctgg acctatggtg agcaagggcg aggaggataa 4620
catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg 4680
ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc 4740
caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca 4800
gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa 4860
gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt 4920
ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct 4980
gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga 5040
ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag 5100
gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa 5160
gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca 5220
caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg 5280
cggcatggac gagctgtaca agtaaggccg cgactctaga gtcgacctgc aggcatgcaa 5340
gcttgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta 5400
tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc 5460
ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga 5520
ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac 5580
ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc 5640
cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc 5700
tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg 5760
gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc 5820
ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc 5880
gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg 5940
ataccgtcga cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 6000
gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 6060
attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 6120
agcaaggggg aggattggga agacaatagc aggcatgctg gggagcttcc tcgctcactg 6180
actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 6240
tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 6300
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 6360
ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 6420
aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 6480
cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 6540
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 6600
aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 6660
cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 6720
ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 6780
gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 6840
gctcttgatc cggcaaacaa accaccgctg gtagcggttt ttttgtttgc aagcagcaga 6900
ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 6960
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 7020
tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 7080
aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 7140
tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 7200
gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 7260
atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 7320
tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 7380
ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 7440
ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 7500
tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 7560
ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 7620
ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 7680
tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 7740
gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 7800
taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 7860
cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 7920
agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 7980
gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 8040
ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc 8093
<210> 26
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> 5518F
<400> 26
ttcgaatccc ctaaaaagaa aagaaaggtg aacatccccg ctctggtgg 49
<210> 27
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> 5518R
<400> 27
ctcggatcca tcggcggtgc gcttcatggt ggcaagctta agtttaaacg cta 53
<210> 28
<211> 8147
<212> DNA
<213> Artificial Sequence
<220>
<223> TC1309: CMV bpNLS-dPspCas13b-Apobec3A(Y132D)序列
<400> 28
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttgcca ccatgaagcg caccgccgat ggatccgagt tcgaatcccc 960
taaaaagaaa agaaaggtga acatccccgc tctggtggaa aaccagaaga agtactttgg 1020
cacctacagc gtgatggcca tgctgaacgc tcagaccgtg ctggaccaca tccagaaggt 1080
ggccgatatt gagggcgagc agaacgagaa caacgagaat ctgtggtttc accccgtgat 1140
gagccacctg tacaacgcca agaacggcta cgacaagcag cccgagaaaa ccatgttcat 1200
catcgagcgg ctgcagagct acttcccatt cctgaagatc atggccgaga accagagaga 1260
gtacagcaac ggcaagtaca agcagaaccg cgtggaagtg aacagcaacg acatcttcga 1320
ggtgctgaag cgcgccttcg gcgtgctgaa gatgtacagg gacctgacca acgcatacaa 1380
gacctacgag gaaaagctga acgacggctg cgagttcctg accagcacag agcaacctct 1440
gagcggcatg atcaacaact actacacagt ggccctgcgg aacatgaacg agagatacgg 1500
ctacaagaca gaggacctgg ccttcatcca ggacaagcgg ttcaagttcg tgaaggacgc 1560
ctacggcaag aaaaagtccc aagtgaatac cggattcttc ctgagcctgc aggactacaa 1620
cggcgacaca cagaagaagc tgcacctgag cggagtggga atcgccctgc tgatctgcct 1680
gttcctggac aagcagtaca tcaacatctt tctgagcagg ctgcccatct tctccagcta 1740
caatgcccag agcgaggaac ggcggatcat catcagatcc ttcggcatca acagcatcaa 1800
gctgcccaag gaccggatcc acagcgagaa gtccaacaag agcgtggcca tggatatgct 1860
caacgaagtg aagcggtgcc ccgacgagct gttcacaaca ctgtctgccg agaagcagtc 1920
ccggttcaga atcatcagcg acgaccacaa tgaagtgctg atgaagcgga gcagcgacag 1980
attcgtgcct ctgctgctgc agtatatcga ttacggcaag ctgttcgacc acatcaggtt 2040
ccacgtgaac atgggcaagc tgagatacct gctgaaggcc gacaagacct gcatcgacgg 2100
ccagaccaga gtcagagtga tcgagcagcc cctgaacggc ttcggcagac tggaagaggc 2160
cgagacaatg cggaagcaag agaacggcac cttcggcaac agcggcatcc ggatcagaga 2220
cttcgagaac atgaagcggg acgacgccaa tcctgccaac tatccctaca tcgtggacac 2280
ctacacacac tacatcctgg aaaacaacaa ggtcgagatg tttatcaacg acaaagagga 2340
cagcgcccca ctgctgcccg tgatcgagga tgatagatac gtggtcaaga caatccccag 2400
ctgccggatg agcaccctgg aaattccagc catggccttc cacatgtttc tgttcggcag 2460
caagaaaacc gagaagctga tcgtggacgt gcacaaccgg tacaagagac tgttccaggc 2520
catgcagaaa gaagaagtga ccgccgagaa tatcgccagc ttcggaatcg ccgagagcga 2580
cctgcctcag aagatcctgg atctgatcag cggcaatgcc cacggcaagg atgtggacgc 2640
cttcatcaga ctgaccgtgg acgacatgct gaccgacacc gagcggagaa tcaagagatt 2700
caaggacgac cggaagtcca ttcggagcgc cgacaacaag atgggaaaga gaggcttcaa 2760
gcagatctcc acaggcaagc tggccgactt cctggccaag gacatcgtgc tgtttcagcc 2820
cagcgtgaac gatggcgaga acaagatcac cggcctgaac taccggatca tgcagagcgc 2880
cattgccgtg tacgatagcg gcgacgatta cgaggccaag cagcagttca agctgatgtt 2940
cgagaaggcc cggctgatcg gcaagggcac aacagagcct catccatttc tgtacaaggt 3000
gttcgcccgc agcatccccg ccaatgccgt cgagttctac gagcgctacc tgatcgagcg 3060
gaagttctac ctgaccggcc tgtccaacga gatcaagaaa ggcaacagag tggatgtgcc 3120
cttcatccgg cgggaccaga acaagtggaa aacacccgcc atgaagaccc tgggcagaat 3180
ctacagcgag gatctgcccg tggaactgcc cagacagatg ttcgacaatg agatcaagtc 3240
ccacctgaag tccctgccac agatggaagg catcgacttc aacaatgcca acgtgaccta 3300
tctgatcgcc gagtacatga agagagtgct ggacgacgac ttccagacct tctaccagtg 3360
gaaccgcaac taccggtaca tggacatgct taagggcgag tacgacagaa agggctccct 3420
gcagcactgc ttcaccagcg tggaagagag agaaggcctc tggaaagagc gggcctccag 3480
aacagagcgg tacagaaagc aggccagcaa caagatccgc agcaaccggc agatgagaaa 3540
cgccagcagc gaagagatcg agacaatcct ggataagcgg ctgagcaaca gccggaacga 3600
gtaccagaaa agcgagaaag tgatccggcg ctacagagtg caggatgccc tgctgtttct 3660
gctggccaaa aagaccctga ccgaactggc cgatttcgac ggcgagaggt tcaaactgaa 3720
agaaatcatg cccgacgccg agaagggaat cctgagcgag atcatgccca tgagcttcac 3780
cttcgagaaa ggcggcaaga agtacaccat caccagcgag ggcatgaagc tgaagaacta 3840
cggcgacttc tttgtgctgg ctagcgacaa gaggatcggc aacctgctgg aactcgtggg 3900
cagcgacatc gtgtccaaag aggatggatc ccttcaactg cctccacttg aaagactgac 3960
actgggatcc ggctcgagca tggaagccag cccagcatcc gggcccagac acttgatgga 4020
tccacacata ttcacttcca actttaacaa tggcattgga aggcataaga cctacctgtg 4080
ctacgaagtg gagcgcctgg acaatggcac ctcggtcaag atggaccagc acaggggctt 4140
tctacacaac caggctaaga atcttctctg tggcttttac ggccgccatg cggagctgcg 4200
cttcttggac ctggttcctt ctttgcagtt ggacccggcc cagatctaca gggtcacttg 4260
gttcatctcc tggagcccct gcttctcctg gggctgtgcc ggggaagtgc gtgcgttcct 4320
tcaggagaac acacacgtga gactgcgtat cttcgctgcc cgcatctatg atgacgaccc 4380
cctatataag gaggcactgc aaatgctgcg ggatgctggg gcccaagtct ccatcatgac 4440
ctacgatgaa tttaagcact gctgggacac ctttgtggac caccagggat gtcccttcca 4500
gccctgggat ggactagatg agcacagcca agccctgagt gggaggctgc gggccattct 4560
ccagaatcag ggaaacgcgg ccgctggatc cgctactaac ttcagcctgc tgaagcaggc 4620
tggagacgtg gaggagaacc ctggacctat ggtgagcaag ggcgaggagg ataacatggc 4680
catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga 4740
gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct 4800
gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat 4860
gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc 4920
cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac 4980
cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg 5040
caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc 5100
ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa 5160
gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc 5220
cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga 5280
ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat 5340
ggacgagctg tacaagtaag gccgcgactc tagagtcgac ctgcaggcat gcaagcttga 5400
taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 5460
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 5520
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 5580
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 5640
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 5700
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 5760
gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 5820
cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 5880
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 5940
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg 6000
tcgacctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 6060
tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 6120
tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 6180
ggggaggatt gggaagacaa tagcaggcat gctggggagc ttcctcgctc actgactcgc 6240
tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 6300
tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 6360
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 6420
agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 6480
accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 6540
ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 6600
gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 6660
ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 6720
gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 6780
taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 6840
tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 6900
gatccggcaa acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc 6960
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 7020
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 7080
agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 7140
ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 7200
gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 7260
catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 7320
cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 7380
cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 7440
gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 7500
tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 7560
gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 7620
tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 7680
gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 7740
gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 7800
taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 7860
tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 7920
ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 7980
taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 8040
tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 8100
aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc 8147
<210> 29
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> 6728F
<400> 29
gtgaacatcc ccgctctggt gg 22
<210> 30
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 6728R
<400> 30
ctttgcagca gctttagggg attcgaactc gga 33
<210> 31
<211> 8147
<212> DNA
<213> Artificial Sequence
<220>
<223> TC1681: CMV-bpNLS(mutant)-dPspCas13b-Apobec3A(Y132D)序列
<400> 31
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttgcca ccatgaagcg caccgccgat ggatccgagt tcgaatcccc 960
taaagctgct gcaaaggtga acatccccgc tctggtggaa aaccagaaga agtactttgg 1020
cacctacagc gtgatggcca tgctgaacgc tcagaccgtg ctggaccaca tccagaaggt 1080
ggccgatatt gagggcgagc agaacgagaa caacgagaat ctgtggtttc accccgtgat 1140
gagccacctg tacaacgcca agaacggcta cgacaagcag cccgagaaaa ccatgttcat 1200
catcgagcgg ctgcagagct acttcccatt cctgaagatc atggccgaga accagagaga 1260
gtacagcaac ggcaagtaca agcagaaccg cgtggaagtg aacagcaacg acatcttcga 1320
ggtgctgaag cgcgccttcg gcgtgctgaa gatgtacagg gacctgacca acgcatacaa 1380
gacctacgag gaaaagctga acgacggctg cgagttcctg accagcacag agcaacctct 1440
gagcggcatg atcaacaact actacacagt ggccctgcgg aacatgaacg agagatacgg 1500
ctacaagaca gaggacctgg ccttcatcca ggacaagcgg ttcaagttcg tgaaggacgc 1560
ctacggcaag aaaaagtccc aagtgaatac cggattcttc ctgagcctgc aggactacaa 1620
cggcgacaca cagaagaagc tgcacctgag cggagtggga atcgccctgc tgatctgcct 1680
gttcctggac aagcagtaca tcaacatctt tctgagcagg ctgcccatct tctccagcta 1740
caatgcccag agcgaggaac ggcggatcat catcagatcc ttcggcatca acagcatcaa 1800
gctgcccaag gaccggatcc acagcgagaa gtccaacaag agcgtggcca tggatatgct 1860
caacgaagtg aagcggtgcc ccgacgagct gttcacaaca ctgtctgccg agaagcagtc 1920
ccggttcaga atcatcagcg acgaccacaa tgaagtgctg atgaagcgga gcagcgacag 1980
attcgtgcct ctgctgctgc agtatatcga ttacggcaag ctgttcgacc acatcaggtt 2040
ccacgtgaac atgggcaagc tgagatacct gctgaaggcc gacaagacct gcatcgacgg 2100
ccagaccaga gtcagagtga tcgagcagcc cctgaacggc ttcggcagac tggaagaggc 2160
cgagacaatg cggaagcaag agaacggcac cttcggcaac agcggcatcc ggatcagaga 2220
cttcgagaac atgaagcggg acgacgccaa tcctgccaac tatccctaca tcgtggacac 2280
ctacacacac tacatcctgg aaaacaacaa ggtcgagatg tttatcaacg acaaagagga 2340
cagcgcccca ctgctgcccg tgatcgagga tgatagatac gtggtcaaga caatccccag 2400
ctgccggatg agcaccctgg aaattccagc catggccttc cacatgtttc tgttcggcag 2460
caagaaaacc gagaagctga tcgtggacgt gcacaaccgg tacaagagac tgttccaggc 2520
catgcagaaa gaagaagtga ccgccgagaa tatcgccagc ttcggaatcg ccgagagcga 2580
cctgcctcag aagatcctgg atctgatcag cggcaatgcc cacggcaagg atgtggacgc 2640
cttcatcaga ctgaccgtgg acgacatgct gaccgacacc gagcggagaa tcaagagatt 2700
caaggacgac cggaagtcca ttcggagcgc cgacaacaag atgggaaaga gaggcttcaa 2760
gcagatctcc acaggcaagc tggccgactt cctggccaag gacatcgtgc tgtttcagcc 2820
cagcgtgaac gatggcgaga acaagatcac cggcctgaac taccggatca tgcagagcgc 2880
cattgccgtg tacgatagcg gcgacgatta cgaggccaag cagcagttca agctgatgtt 2940
cgagaaggcc cggctgatcg gcaagggcac aacagagcct catccatttc tgtacaaggt 3000
gttcgcccgc agcatccccg ccaatgccgt cgagttctac gagcgctacc tgatcgagcg 3060
gaagttctac ctgaccggcc tgtccaacga gatcaagaaa ggcaacagag tggatgtgcc 3120
cttcatccgg cgggaccaga acaagtggaa aacacccgcc atgaagaccc tgggcagaat 3180
ctacagcgag gatctgcccg tggaactgcc cagacagatg ttcgacaatg agatcaagtc 3240
ccacctgaag tccctgccac agatggaagg catcgacttc aacaatgcca acgtgaccta 3300
tctgatcgcc gagtacatga agagagtgct ggacgacgac ttccagacct tctaccagtg 3360
gaaccgcaac taccggtaca tggacatgct taagggcgag tacgacagaa agggctccct 3420
gcagcactgc ttcaccagcg tggaagagag agaaggcctc tggaaagagc gggcctccag 3480
aacagagcgg tacagaaagc aggccagcaa caagatccgc agcaaccggc agatgagaaa 3540
cgccagcagc gaagagatcg agacaatcct ggataagcgg ctgagcaaca gccggaacga 3600
gtaccagaaa agcgagaaag tgatccggcg ctacagagtg caggatgccc tgctgtttct 3660
gctggccaaa aagaccctga ccgaactggc cgatttcgac ggcgagaggt tcaaactgaa 3720
agaaatcatg cccgacgccg agaagggaat cctgagcgag atcatgccca tgagcttcac 3780
cttcgagaaa ggcggcaaga agtacaccat caccagcgag ggcatgaagc tgaagaacta 3840
cggcgacttc tttgtgctgg ctagcgacaa gaggatcggc aacctgctgg aactcgtggg 3900
cagcgacatc gtgtccaaag aggatggatc ccttcaactg cctccacttg aaagactgac 3960
actgggatcc ggctcgagca tggaagccag cccagcatcc gggcccagac acttgatgga 4020
tccacacata ttcacttcca actttaacaa tggcattgga aggcataaga cctacctgtg 4080
ctacgaagtg gagcgcctgg acaatggcac ctcggtcaag atggaccagc acaggggctt 4140
tctacacaac caggctaaga atcttctctg tggcttttac ggccgccatg cggagctgcg 4200
cttcttggac ctggttcctt ctttgcagtt ggacccggcc cagatctaca gggtcacttg 4260
gttcatctcc tggagcccct gcttctcctg gggctgtgcc ggggaagtgc gtgcgttcct 4320
tcaggagaac acacacgtga gactgcgtat cttcgctgcc cgcatctatg atgacgaccc 4380
cctatataag gaggcactgc aaatgctgcg ggatgctggg gcccaagtct ccatcatgac 4440
ctacgatgaa tttaagcact gctgggacac ctttgtggac caccagggat gtcccttcca 4500
gccctgggat ggactagatg agcacagcca agccctgagt gggaggctgc gggccattct 4560
ccagaatcag ggaaacgcgg ccgctggatc cgctactaac ttcagcctgc tgaagcaggc 4620
tggagacgtg gaggagaacc ctggacctat ggtgagcaag ggcgaggagg ataacatggc 4680
catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga 4740
gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct 4800
gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat 4860
gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc 4920
cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac 4980
cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg 5040
caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc 5100
ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa 5160
gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc 5220
cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga 5280
ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat 5340
ggacgagctg tacaagtaag gccgcgactc tagagtcgac ctgcaggcat gcaagcttga 5400
taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 5460
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 5520
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 5580
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 5640
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 5700
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 5760
gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 5820
cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 5880
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 5940
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg 6000
tcgacctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 6060
tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 6120
tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 6180
ggggaggatt gggaagacaa tagcaggcat gctggggagc ttcctcgctc actgactcgc 6240
tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 6300
tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 6360
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 6420
agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 6480
accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 6540
ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 6600
gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 6660
ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 6720
gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 6780
taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 6840
tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 6900
gatccggcaa acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc 6960
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 7020
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 7080
agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 7140
ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 7200
gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 7260
catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 7320
cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 7380
cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 7440
gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 7500
tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 7560
gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 7620
tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 7680
gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 7740
gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 7800
taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 7860
tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 7920
ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 7980
taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 8040
tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 8100
aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc 8147
<210> 32
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> 5666F
<400> 32
ccagaatcag ggaaacgcgg ccggatccaa aagaactgcg gatggatctg agtttgaga 59
<210> 33
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> 5666R
<400> 33
gaagttagta gcggatccga ccttcctctt cttttttgga ctctcaaact cagatccat 59
<210> 34
<211> 8204
<212> DNA
<213> Artificial Sequence
<220>
<223> TC1332: CMV bpNLS-dPspCas13b-Apobec3A(Y132D)-bpNLS序列
<400> 34
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttgcca ccatgaagcg caccgccgat ggatccgagt tcgaatcccc 960
taaaaagaaa agaaaggtga acatccccgc tctggtggaa aaccagaaga agtactttgg 1020
cacctacagc gtgatggcca tgctgaacgc tcagaccgtg ctggaccaca tccagaaggt 1080
ggccgatatt gagggcgagc agaacgagaa caacgagaat ctgtggtttc accccgtgat 1140
gagccacctg tacaacgcca agaacggcta cgacaagcag cccgagaaaa ccatgttcat 1200
catcgagcgg ctgcagagct acttcccatt cctgaagatc atggccgaga accagagaga 1260
gtacagcaac ggcaagtaca agcagaaccg cgtggaagtg aacagcaacg acatcttcga 1320
ggtgctgaag cgcgccttcg gcgtgctgaa gatgtacagg gacctgacca acgcatacaa 1380
gacctacgag gaaaagctga acgacggctg cgagttcctg accagcacag agcaacctct 1440
gagcggcatg atcaacaact actacacagt ggccctgcgg aacatgaacg agagatacgg 1500
ctacaagaca gaggacctgg ccttcatcca ggacaagcgg ttcaagttcg tgaaggacgc 1560
ctacggcaag aaaaagtccc aagtgaatac cggattcttc ctgagcctgc aggactacaa 1620
cggcgacaca cagaagaagc tgcacctgag cggagtggga atcgccctgc tgatctgcct 1680
gttcctggac aagcagtaca tcaacatctt tctgagcagg ctgcccatct tctccagcta 1740
caatgcccag agcgaggaac ggcggatcat catcagatcc ttcggcatca acagcatcaa 1800
gctgcccaag gaccggatcc acagcgagaa gtccaacaag agcgtggcca tggatatgct 1860
caacgaagtg aagcggtgcc ccgacgagct gttcacaaca ctgtctgccg agaagcagtc 1920
ccggttcaga atcatcagcg acgaccacaa tgaagtgctg atgaagcgga gcagcgacag 1980
attcgtgcct ctgctgctgc agtatatcga ttacggcaag ctgttcgacc acatcaggtt 2040
ccacgtgaac atgggcaagc tgagatacct gctgaaggcc gacaagacct gcatcgacgg 2100
ccagaccaga gtcagagtga tcgagcagcc cctgaacggc ttcggcagac tggaagaggc 2160
cgagacaatg cggaagcaag agaacggcac cttcggcaac agcggcatcc ggatcagaga 2220
cttcgagaac atgaagcggg acgacgccaa tcctgccaac tatccctaca tcgtggacac 2280
ctacacacac tacatcctgg aaaacaacaa ggtcgagatg tttatcaacg acaaagagga 2340
cagcgcccca ctgctgcccg tgatcgagga tgatagatac gtggtcaaga caatccccag 2400
ctgccggatg agcaccctgg aaattccagc catggccttc cacatgtttc tgttcggcag 2460
caagaaaacc gagaagctga tcgtggacgt gcacaaccgg tacaagagac tgttccaggc 2520
catgcagaaa gaagaagtga ccgccgagaa tatcgccagc ttcggaatcg ccgagagcga 2580
cctgcctcag aagatcctgg atctgatcag cggcaatgcc cacggcaagg atgtggacgc 2640
cttcatcaga ctgaccgtgg acgacatgct gaccgacacc gagcggagaa tcaagagatt 2700
caaggacgac cggaagtcca ttcggagcgc cgacaacaag atgggaaaga gaggcttcaa 2760
gcagatctcc acaggcaagc tggccgactt cctggccaag gacatcgtgc tgtttcagcc 2820
cagcgtgaac gatggcgaga acaagatcac cggcctgaac taccggatca tgcagagcgc 2880
cattgccgtg tacgatagcg gcgacgatta cgaggccaag cagcagttca agctgatgtt 2940
cgagaaggcc cggctgatcg gcaagggcac aacagagcct catccatttc tgtacaaggt 3000
gttcgcccgc agcatccccg ccaatgccgt cgagttctac gagcgctacc tgatcgagcg 3060
gaagttctac ctgaccggcc tgtccaacga gatcaagaaa ggcaacagag tggatgtgcc 3120
cttcatccgg cgggaccaga acaagtggaa aacacccgcc atgaagaccc tgggcagaat 3180
ctacagcgag gatctgcccg tggaactgcc cagacagatg ttcgacaatg agatcaagtc 3240
ccacctgaag tccctgccac agatggaagg catcgacttc aacaatgcca acgtgaccta 3300
tctgatcgcc gagtacatga agagagtgct ggacgacgac ttccagacct tctaccagtg 3360
gaaccgcaac taccggtaca tggacatgct taagggcgag tacgacagaa agggctccct 3420
gcagcactgc ttcaccagcg tggaagagag agaaggcctc tggaaagagc gggcctccag 3480
aacagagcgg tacagaaagc aggccagcaa caagatccgc agcaaccggc agatgagaaa 3540
cgccagcagc gaagagatcg agacaatcct ggataagcgg ctgagcaaca gccggaacga 3600
gtaccagaaa agcgagaaag tgatccggcg ctacagagtg caggatgccc tgctgtttct 3660
gctggccaaa aagaccctga ccgaactggc cgatttcgac ggcgagaggt tcaaactgaa 3720
agaaatcatg cccgacgccg agaagggaat cctgagcgag atcatgccca tgagcttcac 3780
cttcgagaaa ggcggcaaga agtacaccat caccagcgag ggcatgaagc tgaagaacta 3840
cggcgacttc tttgtgctgg ctagcgacaa gaggatcggc aacctgctgg aactcgtggg 3900
cagcgacatc gtgtccaaag aggatggatc ccttcaactg cctccacttg aaagactgac 3960
actgggatcc ggctcgagca tggaagccag cccagcatcc gggcccagac acttgatgga 4020
tccacacata ttcacttcca actttaacaa tggcattgga aggcataaga cctacctgtg 4080
ctacgaagtg gagcgcctgg acaatggcac ctcggtcaag atggaccagc acaggggctt 4140
tctacacaac caggctaaga atcttctctg tggcttttac ggccgccatg cggagctgcg 4200
cttcttggac ctggttcctt ctttgcagtt ggacccggcc cagatctaca gggtcacttg 4260
gttcatctcc tggagcccct gcttctcctg gggctgtgcc ggggaagtgc gtgcgttcct 4320
tcaggagaac acacacgtga gactgcgtat cttcgctgcc cgcatctatg atgacgaccc 4380
cctatataag gaggcactgc aaatgctgcg ggatgctggg gcccaagtct ccatcatgac 4440
ctacgatgaa tttaagcact gctgggacac ctttgtggac caccagggat gtcccttcca 4500
gccctgggat ggactagatg agcacagcca agccctgagt gggaggctgc gggccattct 4560
ccagaatcag ggaaacgcgg ccggatccaa aagaactgcg gatggatctg agtttgagag 4620
tccaaaaaag aagaggaagg tcggatccgc tactaacttc agcctgctga agcaggctgg 4680
agacgtggag gagaaccctg gacctatggt gagcaagggc gaggaggata acatggccat 4740
catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt 4800
cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa 4860
ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc agttcatgta 4920
cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga agctgtcctt 4980
ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt 5040
gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac 5100
caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg aggcctcctc 5160
cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga ggctgaagct 5220
gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca agaagcccgt 5280
gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc acaacgagga 5340
ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga 5400
cgagctgtac aagtaaggcc gcgactctag agtcgacctg caggcatgca agcttgataa 5460
tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 5520
ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 5580
ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 5640
gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 5700
ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 5760
tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 5820
gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 5880
ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 5940
tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 6000
ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg 6060
acctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 6120
ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 6180
cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 6240
gaggattggg aagacaatag caggcatgct ggggagcttc ctcgctcact gactcgctgc 6300
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 6360
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 6420
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 6480
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 6540
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 6600
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 6660
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 6720
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 6780
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 6840
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 6900
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6960
ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg caagcagcag attacgcgca 7020
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 7080
acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 7140
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 7200
ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 7260
catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 7320
ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 7380
caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 7440
ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 7500
tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 7560
cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 7620
aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 7680
tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 7740
gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 7800
cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 7860
aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 7920
tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 7980
tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 8040
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 8100
atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 8160
taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc 8204
<210> 35
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> 4275Fb
<400> 35
gcggccgctg gatccgctac taacttcagc c 31
<210> 36
<211> 72
<212> DNA
<213> Artificial Sequence
<220>
<223> 4184R
<400> 36
tcttcttggg gctctcaaat tcgctgccgt cagcagtccg tttcatggtg gcaagcttaa 60
gtttaaacgc ta 72
<210> 37
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> 4183F
<400> 37
agagccccaa gaagaagagg aaagtcggat ccatcgagaa gaagaagagc ttcgccaa 58
<210> 38
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 4915R
<400> 38
cttgctggta ccgttggttc ccaggatccg 30
<210> 39
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> 4916F
<400> 39
gaaccaacgg taccagcaag aggacagcag acgggtccga attcgaatcc cccaagaaa 59
<210> 40
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> 4916R
<400> 40
ctggcttcca tagacccggg acccaccttc ctctttttct tgggggattc gaattcgga 59
<210> 41
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> 4915F
<400> 41
gggtctatgg aagccagccc agcatccg 28
<210> 42
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> 4724F
<400> 42
cgcagttctt ttggatccgt ttccctgatt ctggagaatg 40
<210> 43
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> 4874F
<400> 43
ggatccaaaa gaactgcgga tggatct 27
<210> 44
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> 4513R
<400> 44
gatgaagttg cgcatagaac cgaccttcct cttctttttt ggac 44
<210> 45
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> 4514
<400> 45
atgcgcaact tcatcatcaa caacg 25
<210> 46
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> 4183R
<400> 46
ggggattcga actcggatcc atcggcggtg cgcttagaac cggagttgcc gctcacct 58
<210> 47
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> 4858F
<400> 47
tggatccgag ttcgaatccc ctaaaaagaa aagaaaggtg g 41
<210> 48
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> 5802R
<400> 48
agtagcggat ccagcggccg cggatcccac ctttcttttc tttt 44
<210> 49
<211> 4694
<212> DNA
<213> Artificial Sequence
<220>
<223> U6- BbsI- BbsI- PspCas13b DR，crRNA序列
<400> 49
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 60
agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 120
accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 180
ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct 240
gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 300
ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 360
gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 420
taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 480
tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 540
gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 600
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 660
agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 720
cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 780
cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 840
ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 900
taccatctgg ccccagtgct gcaatgatac cgcgggaccc acgctcaccg gctccagatt 960
tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1020
ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1080
atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1140
gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1200
tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1260
cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1320
taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1380
ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1440
ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1500
cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1560
ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 1620
gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 1680
gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 1740
aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg 1800
gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 1860
ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 1920
cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 1980
tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 2040
cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 2100
ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 2160
tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 2220
aaatattaac gtttacaatt tcccattcgc cattcaggct gcgcaactgt tgggaagggc 2280
gatcggtgcg ggcctcttcg ctattacgcc agcccaagct accatgataa gtaagtaata 2340
ttaaggtacg ggaggtactt ggagcggccg caataaaata tctttatttt cattacatct 2400
gtgtgttggt tttttgtgtg aatcgatagt actaacatac gctctccatc aaaacaaaac 2460
gaaacaaaac aaactagcaa aataggctgt ccccagtgca agtgcaggtg ccagaacatt 2520
tctctatcga taggtaccga ttagtgaacg gatctcgacg gtatcgatca cgagactagc 2580
ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata 2640
tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat 2700
tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat 2760
tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg 2820
ctttatatat cttgtggaaa ggacgaaaca ccgaagtctt cgatatcgaa gacttgttgt 2880
ggaaggtcca gttttgaggg gctattacaa ctttttttaa agaattctcg acctcgagac 2940
aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag 3000
gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa 3060
ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca ctttggccgc 3120
ggctcgaggg ggttggggtt gcgccttttc caaggcagcc ctgggtttgc gcagggacgc 3180
ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg accctgggac tcgcacattc 3240
ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta cccttgtggg ccccccggcg 3300
acgcttcctg ctccgcccct aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg 3360
tgacaaacgg aagccgcacg tctcactagt accctcgcag acggacagcg ccagggagca 3420
atggcagcgc gccgaccgcg atgggctgtg gccaatagcg gctgctcagc agggcgcgcc 3480
gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc 3540
ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc 3600
ggctccctcg ttgaccgaat caccgacctc tctccccagg gggatccacc atgaccgagt 3660
acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta cgcaccctcg 3720
ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac cgccacatcg 3780
agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg 3840
tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag 3900
cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg 3960
ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt 4020
tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg 4080
tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg 4140
cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg 4200
tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga gtacctttaa 4260
gaccaatgac ttacaaggca gctgtagatc ttagccactt tctagagtcg gggcggccgg 4320
ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg 4380
cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt 4440
ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag 4500
ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tcgcttcctc 4560
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 4620
ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 4680
aggccagcaa aagg 4694
<210> 50
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 6492T_PGAP2
<400> 50
accgtaagtg agcactagga ggttctccac ga 32
<210> 51
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 6492B_PGAP2
<400> 51
caactcgtgg agaacctcct agtgctcact ta 32
<210> 52
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 6493T_PGAP2
<400> 52
accgcataag tgagcactag gaggttctcc acgaca 36
<210> 53
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 6493B_PGAP2
<400> 53
caactgtcgt ggagaacctc ctagtgctca cttatg 36
<210> 54
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> 6494T_PGAP2
<400> 54
accgaggaga cataagtgag cactaggagg ttctccacga cattgagg 48
<210> 55
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> 6494B_PGAP2
<400> 55
caaccctcaa tgtcgtggag aacctcctag tgctcactta tgtctcct 48
<210> 56
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> 6495T_PGAP2
<400> 56
accgcggagg aggagacata agtgagcact aggaggttct ccacgacatt gaggccgaag 60
<210> 57
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> 6495B_PGAP2
<400> 57
caaccttcgg cctcaatgtc gtggagaacc tcctagtgct cacttatgtc tcctcctccg 60
<210> 58
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 6488T_PGAP2
<400> 58
accgcataag tgagcactgg ttctccacga ca 32
<210> 59
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 6488B_PGAP2
<400> 59
caactcgtgg agaacctcct agtgctcact ta 32
<210> 60
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 6489T_PGAP2
<400> 60
accggacata agtgagcact ggttctccac gacatt 36
<210> 61
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 6489B_PGAP2
<400> 61
caacaatgtc gtggagaacc agtgctcact tatgtc 36
<210> 62
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> 6490T_PGAP2
<400> 62
accgggagga gacataagtg agcactggtt ctccacgaca ttgaggcc 48
<210> 63
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> 6490B_PGAP2
<400> 63
caacggcctc aatgtcgtgg agaaccagtg ctcacttatg tctcctcc 48
<210> 64
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> 6491T_PGAP2
<400> 64
accgctcgga ggaggagaca taagtgagca ctggttctcc acgacattga ggccgaagtt 60
<210> 65
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> 6491B_PGAP2
<400> 65
caacaacttc ggcctcaatg tcgtggagaa ccagtgctca cttatgtctc ctcctccgag 60
<210> 66
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 6496T_PGAP2
<400> 66
accggacata agtgagcatt ctccacgaca tt 32
<210> 67
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 6496B_PGAP2
<400> 67
caacaatgtc gtggagaatg ctcacttatg tc 32
<210> 68
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 6497T_PGAP2
<400> 68
accggagaca taagtgagca ttctccacga cattga 36
<210> 69
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 6497B_PGAP2
<400> 69
caactcaatg tcgtggagaa tgctcactta tgtctc 36
<210> 70
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> 6498T_PGAP2
<400> 70
accggaggag gagacataag tgagcattct ccacgacatt gaggccga 48
<210> 71
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> 6498B_PGAP2
<400> 71
caactcggcc tcaatgtcgt ggagaatgct cacttatgtc tcctcctc 48
<210> 72
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> 6499T_PGAP2
<400> 72
accgtcctcg gaggaggaga cataagtgag cattctccac gacattgagg ccgaagttga 60
<210> 73
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> 6499B_PGAP2
<400> 73
caactcaact tcggcctcaa tgtcgtggag aatgctcact tatgtctcct cctccgagga 60
<210> 74
<211> 4808
<212> DNA
<213> Artificial Sequence
<220>
<223> U6-CasRx crRNA-BspQI- BspQI-CasRx crRNA质粒
<400> 74
cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac 60
gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt 120
cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt 180
ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc 240
gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct 300
ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga 360
ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg 420
cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg 480
gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat 540
gacccgcaag cccggtgcct gagtaccttt aagaccaatg acttacaagg cagctgtaga 600
tcttagccac tttctagagt cggggcggcc ggccgcttcg agcagacatg ataagataca 660
ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 720
tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca 780
acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca 840
agtaaaacct ctacaaatgt ggtcgcttcc tcgctcactg actcgctgcg ctcggtcgtt 900
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 960
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 1020
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 1080
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 1140
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 1200
gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt 1260
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 1320
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 1380
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 1440
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 1500
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 1560
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 1620
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 1680
tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 1740
aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 1800
taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 1860
gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 1920
agtgctgcaa tgataccgcg agatccacgc tcaccggctc cagatttatc agcaataaac 1980
cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 2040
tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 2100
gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 2160
agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 2220
gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 2280
atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 2340
gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 2400
tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 2460
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 2520
agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 2580
gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 2640
cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 2700
tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 2760
ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg 2820
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 2880
cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 2940
aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 3000
cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 3060
ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 3120
aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 3180
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 3240
acaatttccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 3300
tcttcgctat tacgccagcc caagctacca tgataagtaa gtaatattaa ggtacgggag 3360
gtacttggag cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt 3420
tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac 3480
tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgatagg 3540
taccgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg agcggccgcc 3600
cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 3660
gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg 3720
tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 3780
gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg 3840
tggaaaggac gaaacaccgc aagtaaaccc ctaccaactg gtcggggttt gaaacagaag 3900
agcctcgagg ctcttctcaa gtaaacccct accaactggt cggggtttga aacgaagact 3960
ttttttttcg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 4020
gtatcagctc actcaaaggc ggtaatacgg tcctcgagac aaatggcagt attcatccac 4080
aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 4140
atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 4200
cgggtttatt acagggacag cagagatcca ctttggccgc ggctcgaggg ggttggggtt 4260
gcgccttttc caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc 4320
cgggaaacgc agcggcgccg accctgggac tcgcacattc ttcacgtccg ttcgcagcgt 4380
cacccggatc ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct 4440
aagtcgggaa ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg 4500
tctcactagt accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg 4560
atgggctgtg gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg 4620
ggcggtgcgg gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg 4680
ttccgcattc tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat 4740
caccgacctc tctccccagg gggatccacc atgaccgagt acaagcccac ggtgcgcctc 4800
gccacccg 4808
<210> 75
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> 8101T_PGAP2
<400> 75
aaccataagt gagcactagg aggttctcca cgaca 35
<210> 76
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> 8101B_PGAP2
<400> 76
ttgtgtcgtg gagaacctcc tagtgctcac ttatg 35
<210> 77
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> 8102T_PGAP2
<400> 77
aacgacataa gtgagcactg gttctccacg acatt 35
<210> 78
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> 8102B_PGAP2
<400> 78
ttgaatgtcg tggagaacca gtgctcactt atgtc 35
<210> 79
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> 8103T_PGAP2
<400> 79
aacgagacat aagtgagcat tctccacgac attga 35
<210> 80
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> 8103B_PGAP2
<400> 80
ttgtcaatgt cgtggagaat gctcacttat gtctc 35
<210> 81
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> 8118T_PGAP2
<400> 81
aacgacataa gtgagcattc tccacgacat t 31
<210> 82
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> 8118B_PGAP2
<400> 82
ttgaatgtcg tggagaatgc tcacttatgt c 31
<210> 83
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR引物
<400> 83
gccacaagct ggagtacaac 20
<210> 84
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR引物
<400> 84
tagagaacct ccctgtcagg ta 22
<210> 85
<211> 199
<212> PRT
<213> Homo sapiens
<400> 85
Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His
1 5 10 15
Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr
20 25 30
Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met
35 40 45
Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys
50 55 60
Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro
65 70 75 80
Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile
85 90 95
Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala
100 105 110
Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg
115 120 125
Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg
130 135 140
Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His
145 150 155 160
Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp
165 170 175
Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala
180 185 190
Ile Leu Gln Asn Gln Gly Asn
195
<210> 86
<211> 1433
<212> DNA
<213> Artificial Sequence
<220>
<223> TC243上的mcherry和WPRE片段
<400> 86
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctatg 60
gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg 120
cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc 180
ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc 240
gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc 300
gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg 360
atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc 420
gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg 480
cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc 540
ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag 600
gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac 660
atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc 720
gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaagg ccgcgactct 780
agagtcgacc tgcaggcatg caagcttgat atcaagctta tcgataatca acctctggat 840
tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 900
ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 960
tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 1020
caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 1080
accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 1140
ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 1200
tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc 1260
tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 1320
ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 1380
acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc tcg 1433
<210> 87
<211> 817
<212> DNA
<213> Artificial Sequence
<220>
<223> TC332上的WPRE和PolyA片段
<400> 87
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240
ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300
attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360
ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420
gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt 600
cgacctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 660
ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 720
cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 780
gggaggattg ggaagacaat agcaggcatg ctgggga 817
<210> 88
<211> 1709
<212> DNA
<213> Artificial Sequence
<220>
<223> 以TC1316扩增出的第一dCas13Rx片段
<400> 88
agagccccaa gaagaagagg aaagtcggat ccatcgagaa gaagaagagc ttcgccaagg 60
gcatgggagt gaagagcacc ctggtgtccg gctctaaggt gtacatgacc acatttgctg 120
agggaagcga cgccaggctg gagaagatcg tggagggcga tagcatcaga tccgtgaacg 180
agggagaggc tttcagcgcc gagatggctg acaagaacgc tggctacaag atcggaaacg 240
ccaagttttc ccacccaaag ggctacgccg tggtggctaa caacccactg tacaccggac 300
cagtgcagca ggacatgctg ggactgaagg agacactgga gaagaggtac ttcggcgagt 360
ccgccgacgg aaacgataac atctgcatcc aggtcatcca caacatcctg gatatcgaga 420
agatcctggc tgagtacatc acaaacgccg cttacgccgt gaacaacatc tccggcctgg 480
acaaggatat catcggcttc ggaaagtttt ctaccgtgta cacatacgac gagttcaagg 540
atccagagca ccaccgggcc gcttttaaca acaacgacaa gctgatcaac gccatcaagg 600
ctcagtacga cgagttcgat aactttctgg ataaccccag gctgggctac ttcggacagg 660
ctttcttttc taaggagggc agaaactaca tcatcaacta cggaaacgag tgttacgaca 720
tcctggccct gctgagcgga ctggcccact gggtggtggc caacaacgag gaggagtctc 780
ggatcagccg cacctggctg tacaacctgg acaagaacct ggataacgag tacatctcca 840
cactgaacta cctgtacgac aggatcacca acgagctgac aaacagcttc tccaagaact 900
ctgccgctaa cgtgaactac atcgctgaga ccctgggcat caacccagct gagttcgctg 960
agcagtactt cagattttcc atcatgaagg agcagaagaa cctgggcttc aacatcacaa 1020
agctgagaga agtgatgctg gacagaaagg atatgtccga gatcaggaag aaccacaagg 1080
tgttcgattc tatcagaacc aaggtgtaca caatgatgga ctttgtgatc tacaggtact 1140
acatcgagga ggatgccaag gtggccgctg ccaacaagag cctgcccgac aacgagaagt 1200
ctctgagcga gaaggatatc ttcgtgatca acctgagagg ctcctttaac gacgatcaga 1260
aggacgctct gtactacgat gaggccaaca ggatctggag aaagctggag aacatcatgc 1320
acaacatcaa ggagttccgg ggaaacaaga cccgcgagta caagaagaag gacgctccaa 1380
ggctgcctag gatcctgcct gctggaaggg acgtgagcgc cttcagcaag ctgatgtacg 1440
ccctgacaat gtttctggac ggaaaggaga tcaacgatct gctgaccaca ctgatcaaca 1500
agttcgacaa catccagtct tttctgaaag tgatgcctct gatcggcgtg aacgctaagt 1560
tcgtggagga gtacgccttc tttaaggaca gcgccaagat cgctgatgag ctgcggctga 1620
tcaagtcctt tgccaggatg ggagagccaa tcgctgacgc taggagagct atgtacatcg 1680
atgccatccg gatcctggga accaacggt 1709
<210> 89
<211> 1290
<212> DNA
<213> Artificial Sequence
<220>
<223> 荧光报告系统质粒-第一质粒
<400> 89
gaagtaagtg ctcttgaaaa agaggtgtct gcccttgaaa aggaagtctc agcactggaa 60
aaggaggtat ccgcacttga gaaggaggta agtgccctgg agaagggcgg cagcggcggc 120
atgtccaagg gagaggagct gttcaccgga gtggtgccaa tcctggtgga gctggacggc 180
gatgtgaacg gccacaagtt ttctgtgcgc ggagagggag agggcgacgc aaccatcggc 240
aagctgacac tgaagttcat ctgcaccaca ggcaagctgc ccgtgccttg gccaaccctg 300
gtgaccacac tgacatacgg cgtgcagtgt ttctctaggt atccagacca catgaagaga 360
cacgatttct ttaagagcgc catgccagag ggatacgtgc aggagcggac catctccttc 420
aaggacgatg gcaagtataa gacccgcgcc gtggtgaagt ttgagggcga tacactggtg 480
aacaggatcg agctgaaggg caccgacttc aaggaggatg gcaatatcct gggccacaag 540
ctggagtaca actttaatag ccacaacgtg tatatcacag ccgacaagca gaagaacggc 600
atcaaggcca acttcaccgt gaggcacaat gtggaggacg gctccgtgca gctggccgat 660
cactatcaac aaaacacacc tatcggcgac ggacccgtgc tgctgcctga taatcactat 720
ctgtccaccc aaaccgtgct gtctaaggac ccaaacgaga aggcggccgc tacttgccac 780
cgacgaccaa aatatcgagc agacaccgtg aggataaatt attcgaatat tacgaccaac 840
ggagcggccg ctaagcgcac cgccgatggc tccgagttcg aatcccctaa aaagaaaaga 900
aaggtgggag gaggatctgg cgggaaggtt agcgcactga aggaaaaagt ttcagcactg 960
aaagagaagg tgagtgccct caaagagaaa gtctccgctc tcaaggaaaa agtctcagcc 1020
cttaaagaga gaaaggtggg aggaggatcc ggcgggaact cctctccacc agcagtcacc 1080
ctgacgcacc caatcaccaa aatcgatacc aaatacatca tgacatgcat gtcggccgac 1140
ctggaggtcg tcacgagcac ctgggtgctc gttggcggcg tcctggctgc tctggccgcg 1200
tattgcctgt caacaggctg cgtggtcata gtgggcagga tcgtcttgtc cgggaagccg 1260
gcaattatac ctgacaggga ggttctctag 1290
<210> 90
<211> 199
<212> PRT
<213> Artificial Sequence
<220>
<223> APOBEC3A(H29K)片段
<400> 90
Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His
1 5 10 15
Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg Lys Lys Thr Tyr
20 25 30
Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met
35 40 45
Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys
50 55 60
Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro
65 70 75 80
Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile
85 90 95
Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala
100 105 110
Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg
115 120 125
Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg
130 135 140
Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His
145 150 155 160
Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp
165 170 175
Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala
180 185 190
Ile Leu Gln Asn Gln Gly Asn
195
<210> 91
<211> 966
<212> PRT
<213> Ruminococcus flavefaciensstrain XPD3002
<400> 91
Ile Glu Lys Lys Lys Ser Phe Ala Lys Gly Met Gly Val Lys Ser Thr
1 5 10 15
Leu Val Ser Gly Ser Lys Val Tyr Met Thr Thr Phe Ala Glu Gly Ser
20 25 30
Asp Ala Arg Leu Glu Lys Ile Val Glu Gly Asp Ser Ile Arg Ser Val
35 40 45
Asn Glu Gly Glu Ala Phe Ser Ala Glu Met Ala Asp Lys Asn Ala Gly
50 55 60
Tyr Lys Ile Gly Asn Ala Lys Phe Ser His Pro Lys Gly Tyr Ala Val
65 70 75 80
Val Ala Asn Asn Pro Leu Tyr Thr Gly Pro Val Gln Gln Asp Met Leu
85 90 95
Gly Leu Lys Glu Thr Leu Glu Lys Arg Tyr Phe Gly Glu Ser Ala Asp
100 105 110
Gly Asn Asp Asn Ile Cys Ile Gln Val Ile His Asn Ile Leu Asp Ile
115 120 125
Glu Lys Ile Leu Ala Glu Tyr Ile Thr Asn Ala Ala Tyr Ala Val Asn
130 135 140
Asn Ile Ser Gly Leu Asp Lys Asp Ile Ile Gly Phe Gly Lys Phe Ser
145 150 155 160
Thr Val Tyr Thr Tyr Asp Glu Phe Lys Asp Pro Glu His His Arg Ala
165 170 175
Ala Phe Asn Asn Asn Asp Lys Leu Ile Asn Ala Ile Lys Ala Gln Tyr
180 185 190
Asp Glu Phe Asp Asn Phe Leu Asp Asn Pro Arg Leu Gly Tyr Phe Gly
195 200 205
Gln Ala Phe Phe Ser Lys Glu Gly Arg Asn Tyr Ile Ile Asn Tyr Gly
210 215 220
Asn Glu Cys Tyr Asp Ile Leu Ala Leu Leu Ser Gly Leu Ala His Trp
225 230 235 240
Val Val Ala Asn Asn Glu Glu Glu Ser Arg Ile Ser Arg Thr Trp Leu
245 250 255
Tyr Asn Leu Asp Lys Asn Leu Asp Asn Glu Tyr Ile Ser Thr Leu Asn
260 265 270
Tyr Leu Tyr Asp Arg Ile Thr Asn Glu Leu Thr Asn Ser Phe Ser Lys
275 280 285
Asn Ser Ala Ala Asn Val Asn Tyr Ile Ala Glu Thr Leu Gly Ile Asn
290 295 300
Pro Ala Glu Phe Ala Glu Gln Tyr Phe Arg Phe Ser Ile Met Lys Glu
305 310 315 320
Gln Lys Asn Leu Gly Phe Asn Ile Thr Lys Leu Arg Glu Val Met Leu
325 330 335
Asp Arg Lys Asp Met Ser Glu Ile Arg Lys Asn His Lys Val Phe Asp
340 345 350
Ser Ile Arg Thr Lys Val Tyr Thr Met Met Asp Phe Val Ile Tyr Arg
355 360 365
Tyr Tyr Ile Glu Glu Asp Ala Lys Val Ala Ala Ala Asn Lys Ser Leu
370 375 380
Pro Asp Asn Glu Lys Ser Leu Ser Glu Lys Asp Ile Phe Val Ile Asn
385 390 395 400
Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys Asp Ala Leu Tyr Tyr Asp
405 410 415
Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu Asn Ile Met His Asn Ile
420 425 430
Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu Tyr Lys Lys Lys Asp Ala
435 440 445
Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly Arg Asp Val Ser Ala Phe
450 455 460
Ser Lys Leu Met Tyr Ala Leu Thr Met Phe Leu Asp Gly Lys Glu Ile
465 470 475 480
Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe Asp Asn Ile Gln Ser
485 490 495
Phe Leu Lys Val Met Pro Leu Ile Gly Val Asn Ala Lys Phe Val Glu
500 505 510
Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys Ile Ala Asp Glu Leu Arg
515 520 525
Leu Ile Lys Ser Phe Ala Arg Met Gly Glu Pro Ile Ala Asp Ala Arg
530 535 540
Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile Leu Gly Thr Asn Leu Ser
545 550 555 560
Tyr Asp Glu Leu Lys Ala Leu Ala Asp Thr Phe Ser Leu Asp Glu Asn
565 570 575
Gly Asn Lys Leu Lys Lys Gly Lys His Gly Met Arg Asn Phe Ile Ile
580 585 590
Asn Asn Val Ile Ser Asn Lys Arg Phe His Tyr Leu Ile Arg Tyr Gly
595 600 605
Asp Pro Ala His Leu His Glu Ile Ala Lys Asn Glu Ala Val Val Lys
610 615 620
Phe Val Leu Gly Arg Ile Ala Asp Ile Gln Lys Lys Gln Gly Gln Asn
625 630 635 640
Gly Lys Asn Gln Ile Asp Arg Tyr Tyr Glu Thr Cys Ile Gly Lys Asp
645 650 655
Lys Gly Lys Ser Val Ser Glu Lys Val Asp Ala Leu Thr Lys Ile Ile
660 665 670
Thr Gly Met Asn Tyr Asp Gln Phe Asp Lys Lys Arg Ser Val Ile Glu
675 680 685
Asp Thr Gly Arg Glu Asn Ala Glu Arg Glu Lys Phe Lys Lys Ile Ile
690 695 700
Ser Leu Tyr Leu Thr Val Ile Tyr His Ile Leu Lys Asn Ile Val Asn
705 710 715 720
Ile Asn Ala Arg Tyr Val Ile Gly Phe His Cys Val Glu Arg Asp Ala
725 730 735
Gln Leu Tyr Lys Glu Lys Gly Tyr Asp Ile Asn Leu Lys Lys Leu Glu
740 745 750
Glu Lys Gly Phe Ser Ser Val Thr Lys Leu Cys Ala Gly Ile Asp Glu
755 760 765
Thr Ala Pro Asp Lys Arg Lys Asp Val Glu Lys Glu Met Ala Glu Arg
770 775 780
Ala Lys Glu Ser Ile Asp Ser Leu Glu Ser Ala Asn Pro Lys Leu Tyr
785 790 795 800
Ala Asn Tyr Ile Lys Tyr Ser Asp Glu Lys Lys Ala Glu Glu Phe Thr
805 810 815
Arg Gln Ile Asn Arg Glu Lys Ala Lys Thr Ala Leu Asn Ala Tyr Leu
820 825 830
Arg Asn Thr Lys Trp Asn Val Ile Ile Arg Glu Asp Leu Leu Arg Ile
835 840 845
Asp Asn Lys Thr Cys Thr Leu Phe Ala Asn Lys Ala Val Ala Leu Glu
850 855 860
Val Ala Arg Tyr Val His Ala Tyr Ile Asn Asp Ile Ala Glu Val Asn
865 870 875 880
Ser Tyr Phe Gln Leu Tyr His Tyr Ile Met Gln Arg Ile Ile Met Asn
885 890 895
Glu Arg Tyr Glu Lys Ser Ser Gly Lys Val Ser Glu Tyr Phe Asp Ala
900 905 910
Val Asn Asp Glu Lys Lys Tyr Asn Asp Arg Leu Leu Lys Leu Leu Cys
915 920 925
Val Pro Phe Gly Tyr Cys Ile Pro Arg Phe Lys Asn Leu Ser Ile Glu
930 935 940
Ala Leu Phe Asp Arg Asn Glu Ala Ala Lys Phe Asp Lys Glu Lys Lys
945 950 955 960
Lys Val Ser Gly Asn Ser
965
<210> 92
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> sgRNA
<400> 92
ttatcctcac ggtgtcgtcg tcggtggcaa gt 32
<210> 93
<211> 1295
<212> PRT
<213> Artificial Sequence
<220>
<223> CURE-X1
<400> 93
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val Gly Ser Ile Glu Lys Lys Lys Ser Phe Ala Lys Gly Met Gly
20 25 30
Val Lys Ser Thr Leu Val Ser Gly Ser Lys Val Tyr Met Thr Thr Phe
35 40 45
Ala Glu Gly Ser Asp Ala Arg Leu Glu Lys Ile Val Glu Gly Asp Ser
50 55 60
Ile Arg Ser Val Asn Glu Gly Glu Ala Phe Ser Ala Glu Met Ala Asp
65 70 75 80
Lys Asn Ala Gly Tyr Lys Ile Gly Asn Ala Lys Phe Ser His Pro Lys
85 90 95
Gly Tyr Ala Val Val Ala Asn Asn Pro Leu Tyr Thr Gly Pro Val Gln
100 105 110
Gln Asp Met Leu Gly Leu Lys Glu Thr Leu Glu Lys Arg Tyr Phe Gly
115 120 125
Glu Ser Ala Asp Gly Asn Asp Asn Ile Cys Ile Gln Val Ile His Asn
130 135 140
Ile Leu Asp Ile Glu Lys Ile Leu Ala Glu Tyr Ile Thr Asn Ala Ala
145 150 155 160
Tyr Ala Val Asn Asn Ile Ser Gly Leu Asp Lys Asp Ile Ile Gly Phe
165 170 175
Gly Lys Phe Ser Thr Val Tyr Thr Tyr Asp Glu Phe Lys Asp Pro Glu
180 185 190
His His Arg Ala Ala Phe Asn Asn Asn Asp Lys Leu Gly Ser Asn Ser
195 200 205
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
210 215 220
Lys Val Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
225 230 235 240
Ser Gly Gly Ser Ala Ala Ala Gly Ser Met Glu Ala Ser Pro Ala Ser
245 250 255
Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser Asn Phe Asn
260 265 270
Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr Glu Val Glu Arg
275 280 285
Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln His Arg Gly Phe Leu
290 295 300
His Asn Gln Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly Arg His Ala
305 310 315 320
Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu Gln Leu Asp Pro Ala
325 330 335
Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys Phe Ser
340 345 350
Trp Gly Cys Ala Gly Glu Val Arg Ala Phe Leu Gln Glu Asn Thr His
355 360 365
Val Arg Leu Arg Ile Phe Ala Ala Arg Ile Tyr Asp Asp Asp Pro Leu
370 375 380
Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser
385 390 395 400
Ile Met Thr Tyr Asp Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp
405 410 415
His Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser
420 425 430
Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn
435 440 445
Ala Ala Ala Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
450 455 460
Ser Gly Gly Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe
465 470 475 480
Glu Ser Pro Lys Lys Lys Arg Lys Val Gly Pro Gly Ser Ile Asn Ala
485 490 495
Ile Lys Ala Gln Tyr Asp Glu Phe Asp Asn Phe Leu Asp Asn Pro Arg
500 505 510
Leu Gly Tyr Phe Gly Gln Ala Phe Phe Ser Lys Glu Gly Arg Asn Tyr
515 520 525
Ile Ile Asn Tyr Gly Asn Glu Cys Tyr Asp Ile Leu Ala Leu Leu Ser
530 535 540
Gly Leu Ala His Trp Val Val Ala Asn Asn Glu Glu Glu Ser Arg Ile
545 550 555 560
Ser Arg Thr Trp Leu Tyr Asn Leu Asp Lys Asn Leu Asp Asn Glu Tyr
565 570 575
Ile Ser Thr Leu Asn Tyr Leu Tyr Asp Arg Ile Thr Asn Glu Leu Thr
580 585 590
Asn Ser Phe Ser Lys Asn Ser Ala Ala Asn Val Asn Tyr Ile Ala Glu
595 600 605
Thr Leu Gly Ile Asn Pro Ala Glu Phe Ala Glu Gln Tyr Phe Arg Phe
610 615 620
Ser Ile Met Lys Glu Gln Lys Asn Leu Gly Phe Asn Ile Thr Lys Leu
625 630 635 640
Arg Glu Val Met Leu Asp Arg Lys Asp Met Ser Glu Ile Arg Lys Asn
645 650 655
His Lys Val Phe Asp Ser Ile Arg Thr Lys Val Tyr Thr Met Met Asp
660 665 670
Phe Val Ile Tyr Arg Tyr Tyr Ile Glu Glu Asp Ala Lys Val Ala Ala
675 680 685
Ala Asn Lys Ser Leu Pro Asp Asn Glu Lys Ser Leu Ser Glu Lys Asp
690 695 700
Ile Phe Val Ile Asn Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys Asp
705 710 715 720
Ala Leu Tyr Tyr Asp Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu Asn
725 730 735
Ile Met His Asn Ile Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu Tyr
740 745 750
Lys Lys Lys Asp Ala Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly Arg
755 760 765
Asp Val Ser Ala Phe Ser Lys Leu Met Tyr Ala Leu Thr Met Phe Leu
770 775 780
Asp Gly Lys Glu Ile Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe
785 790 795 800
Asp Asn Ile Gln Ser Phe Leu Lys Val Met Pro Leu Ile Gly Val Asn
805 810 815
Ala Lys Phe Val Glu Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys Ile
820 825 830
Ala Asp Glu Leu Arg Leu Ile Lys Ser Phe Ala Arg Met Gly Glu Pro
835 840 845
Ile Ala Asp Ala Arg Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile Leu
850 855 860
Gly Thr Asn Leu Ser Tyr Asp Glu Leu Lys Ala Leu Ala Asp Thr Phe
865 870 875 880
Ser Leu Asp Glu Asn Gly Asn Lys Leu Lys Lys Gly Lys His Gly Met
885 890 895
Arg Asn Phe Ile Ile Asn Asn Val Ile Ser Asn Lys Arg Phe His Tyr
900 905 910
Leu Ile Arg Tyr Gly Asp Pro Ala His Leu His Glu Ile Ala Lys Asn
915 920 925
Glu Ala Val Val Lys Phe Val Leu Gly Arg Ile Ala Asp Ile Gln Lys
930 935 940
Lys Gln Gly Gln Asn Gly Lys Asn Gln Ile Asp Arg Tyr Tyr Glu Thr
945 950 955 960
Cys Ile Gly Lys Asp Lys Gly Lys Ser Val Ser Glu Lys Val Asp Ala
965 970 975
Leu Thr Lys Ile Ile Thr Gly Met Asn Tyr Asp Gln Phe Asp Lys Lys
980 985 990
Arg Ser Val Ile Glu Asp Thr Gly Arg Glu Asn Ala Glu Arg Glu Lys
995 1000 1005
Phe Lys Lys Ile Ile Ser Leu Tyr Leu Thr Val Ile Tyr His Ile
1010 1015 1020
Leu Lys Asn Ile Val Asn Ile Asn Ala Arg Tyr Val Ile Gly Phe
1025 1030 1035
His Cys Val Glu Arg Asp Ala Gln Leu Tyr Lys Glu Lys Gly Tyr
1040 1045 1050
Asp Ile Asn Leu Lys Lys Leu Glu Glu Lys Gly Phe Ser Ser Val
1055 1060 1065
Thr Lys Leu Cys Ala Gly Ile Asp Glu Thr Ala Pro Asp Lys Arg
1070 1075 1080
Lys Asp Val Glu Lys Glu Met Ala Glu Arg Ala Lys Glu Ser Ile
1085 1090 1095
Asp Ser Leu Glu Ser Ala Asn Pro Lys Leu Tyr Ala Asn Tyr Ile
1100 1105 1110
Lys Tyr Ser Asp Glu Lys Lys Ala Glu Glu Phe Thr Arg Gln Ile
1115 1120 1125
Asn Arg Glu Lys Ala Lys Thr Ala Leu Asn Ala Tyr Leu Arg Asn
1130 1135 1140
Thr Lys Trp Asn Val Ile Ile Arg Glu Asp Leu Leu Arg Ile Asp
1145 1150 1155
Asn Lys Thr Cys Thr Leu Phe Ala Asn Lys Ala Val Ala Leu Glu
1160 1165 1170
Val Ala Arg Tyr Val His Ala Tyr Ile Asn Asp Ile Ala Glu Val
1175 1180 1185
Asn Ser Tyr Phe Gln Leu Tyr His Tyr Ile Met Gln Arg Ile Ile
1190 1195 1200
Met Asn Glu Arg Tyr Glu Lys Ser Ser Gly Lys Val Ser Glu Tyr
1205 1210 1215
Phe Asp Ala Val Asn Asp Glu Lys Lys Tyr Asn Asp Arg Leu Leu
1220 1225 1230
Lys Leu Leu Cys Val Pro Phe Gly Tyr Cys Ile Pro Arg Phe Lys
1235 1240 1245
Asn Leu Ser Ile Glu Ala Leu Phe Asp Arg Asn Glu Ala Ala Lys
1250 1255 1260
Phe Asp Lys Glu Lys Lys Lys Val Ser Gly Asn Ser Gly Ser Lys
1265 1270 1275
Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1280 1285 1290
Lys Val
1295
<210> 94
<211> 1280
<212> PRT
<213> Artificial Sequence
<220>
<223> CURE-X2
<400> 94
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val Gly Ser Ile Glu Lys Lys Lys Ser Phe Ala Lys Gly Met Gly
20 25 30
Val Lys Ser Thr Leu Val Ser Gly Ser Lys Val Tyr Met Thr Thr Phe
35 40 45
Ala Glu Gly Ser Asp Ala Arg Leu Glu Lys Ile Val Glu Gly Asp Ser
50 55 60
Ile Arg Ser Val Asn Glu Gly Glu Ala Phe Ser Ala Glu Met Ala Asp
65 70 75 80
Lys Asn Ala Gly Tyr Lys Ile Gly Asn Ala Lys Phe Ser His Pro Lys
85 90 95
Gly Tyr Ala Val Val Ala Asn Asn Pro Leu Tyr Thr Gly Pro Val Gln
100 105 110
Gln Asp Met Leu Gly Leu Lys Glu Thr Leu Glu Lys Arg Tyr Phe Gly
115 120 125
Glu Ser Ala Asp Gly Asn Asp Asn Ile Cys Ile Gln Val Ile His Asn
130 135 140
Ile Leu Asp Ile Glu Lys Ile Leu Ala Glu Tyr Ile Thr Asn Ala Ala
145 150 155 160
Tyr Ala Val Asn Asn Ile Ser Gly Leu Asp Lys Asp Ile Ile Gly Phe
165 170 175
Gly Lys Phe Ser Thr Val Tyr Thr Tyr Asp Glu Phe Lys Asp Pro Glu
180 185 190
His His Arg Ala Ala Phe Asn Asn Asn Asp Lys Leu Ile Asn Ala Ile
195 200 205
Lys Ala Gln Tyr Asp Glu Phe Asp Asn Phe Leu Asp Asn Pro Arg Leu
210 215 220
Gly Tyr Phe Gly Gln Ala Phe Phe Ser Lys Glu Gly Arg Asn Tyr Ile
225 230 235 240
Ile Asn Tyr Gly Asn Glu Cys Tyr Asp Ile Leu Ala Leu Leu Ser Gly
245 250 255
Leu Ala His Trp Val Val Ala Asn Asn Glu Glu Glu Ser Arg Ile Ser
260 265 270
Arg Thr Trp Leu Tyr Asn Leu Asp Lys Asn Leu Asp Asn Glu Tyr Ile
275 280 285
Ser Thr Leu Asn Tyr Leu Tyr Asp Arg Ile Thr Asn Glu Leu Thr Asn
290 295 300
Ser Phe Ser Lys Asn Ser Ala Ala Asn Val Asn Tyr Ile Ala Glu Thr
305 310 315 320
Leu Gly Ile Asn Pro Ala Glu Phe Ala Glu Gln Tyr Phe Arg Phe Ser
325 330 335
Ile Met Lys Glu Gln Lys Asn Leu Gly Phe Asn Ile Thr Lys Leu Arg
340 345 350
Glu Val Met Leu Asp Arg Lys Asp Met Ser Glu Ile Arg Lys Asn His
355 360 365
Lys Val Phe Asp Ser Ile Arg Thr Lys Val Tyr Thr Met Met Asp Phe
370 375 380
Val Ile Tyr Arg Tyr Tyr Ile Glu Glu Asp Ala Lys Gly Ser Asn Ser
385 390 395 400
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
405 410 415
Lys Val Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
420 425 430
Ser Gly Gly Ser Ala Ala Ala Gly Ser Met Glu Ala Ser Pro Ala Ser
435 440 445
Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser Asn Phe Asn
450 455 460
Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr Glu Val Glu Arg
465 470 475 480
Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln His Arg Gly Phe Leu
485 490 495
His Asn Gln Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly Arg His Ala
500 505 510
Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu Gln Leu Asp Pro Ala
515 520 525
Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys Phe Ser
530 535 540
Trp Gly Cys Ala Gly Glu Val Arg Ala Phe Leu Gln Glu Asn Thr His
545 550 555 560
Val Arg Leu Arg Ile Phe Ala Ala Arg Ile Tyr Asp Asp Asp Pro Leu
565 570 575
Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser
580 585 590
Ile Met Thr Tyr Asp Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp
595 600 605
His Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser
610 615 620
Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn
625 630 635 640
Ala Ala Ala Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
645 650 655
Ser Gly Gly Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe
660 665 670
Glu Ser Pro Lys Lys Lys Arg Lys Val Gly Pro Gly Ser Ser Glu Lys
675 680 685
Asp Ile Phe Val Ile Asn Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys
690 695 700
Asp Ala Leu Tyr Tyr Asp Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu
705 710 715 720
Asn Ile Met His Asn Ile Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu
725 730 735
Tyr Lys Lys Lys Asp Ala Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly
740 745 750
Arg Asp Val Ser Ala Phe Ser Lys Leu Met Tyr Ala Leu Thr Met Phe
755 760 765
Leu Asp Gly Lys Glu Ile Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys
770 775 780
Phe Asp Asn Ile Gln Ser Phe Leu Lys Val Met Pro Leu Ile Gly Val
785 790 795 800
Asn Ala Lys Phe Val Glu Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys
805 810 815
Ile Ala Asp Glu Leu Arg Leu Ile Lys Ser Phe Ala Arg Met Gly Glu
820 825 830
Pro Ile Ala Asp Ala Arg Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile
835 840 845
Leu Gly Thr Asn Leu Ser Tyr Asp Glu Leu Lys Ala Leu Ala Asp Thr
850 855 860
Phe Ser Leu Asp Glu Asn Gly Asn Lys Leu Lys Lys Gly Lys His Gly
865 870 875 880
Met Arg Asn Phe Ile Ile Asn Asn Val Ile Ser Asn Lys Arg Phe His
885 890 895
Tyr Leu Ile Arg Tyr Gly Asp Pro Ala His Leu His Glu Ile Ala Lys
900 905 910
Asn Glu Ala Val Val Lys Phe Val Leu Gly Arg Ile Ala Asp Ile Gln
915 920 925
Lys Lys Gln Gly Gln Asn Gly Lys Asn Gln Ile Asp Arg Tyr Tyr Glu
930 935 940
Thr Cys Ile Gly Lys Asp Lys Gly Lys Ser Val Ser Glu Lys Val Asp
945 950 955 960
Ala Leu Thr Lys Ile Ile Thr Gly Met Asn Tyr Asp Gln Phe Asp Lys
965 970 975
Lys Arg Ser Val Ile Glu Asp Thr Gly Arg Glu Asn Ala Glu Arg Glu
980 985 990
Lys Phe Lys Lys Ile Ile Ser Leu Tyr Leu Thr Val Ile Tyr His Ile
995 1000 1005
Leu Lys Asn Ile Val Asn Ile Asn Ala Arg Tyr Val Ile Gly Phe
1010 1015 1020
His Cys Val Glu Arg Asp Ala Gln Leu Tyr Lys Glu Lys Gly Tyr
1025 1030 1035
Asp Ile Asn Leu Lys Lys Leu Glu Glu Lys Gly Phe Ser Ser Val
1040 1045 1050
Thr Lys Leu Cys Ala Gly Ile Asp Glu Thr Ala Pro Asp Lys Arg
1055 1060 1065
Lys Asp Val Glu Lys Glu Met Ala Glu Arg Ala Lys Glu Ser Ile
1070 1075 1080
Asp Ser Leu Glu Ser Ala Asn Pro Lys Leu Tyr Ala Asn Tyr Ile
1085 1090 1095
Lys Tyr Ser Asp Glu Lys Lys Ala Glu Glu Phe Thr Arg Gln Ile
1100 1105 1110
Asn Arg Glu Lys Ala Lys Thr Ala Leu Asn Ala Tyr Leu Arg Asn
1115 1120 1125
Thr Lys Trp Asn Val Ile Ile Arg Glu Asp Leu Leu Arg Ile Asp
1130 1135 1140
Asn Lys Thr Cys Thr Leu Phe Ala Asn Lys Ala Val Ala Leu Glu
1145 1150 1155
Val Ala Arg Tyr Val His Ala Tyr Ile Asn Asp Ile Ala Glu Val
1160 1165 1170
Asn Ser Tyr Phe Gln Leu Tyr His Tyr Ile Met Gln Arg Ile Ile
1175 1180 1185
Met Asn Glu Arg Tyr Glu Lys Ser Ser Gly Lys Val Ser Glu Tyr
1190 1195 1200
Phe Asp Ala Val Asn Asp Glu Lys Lys Tyr Asn Asp Arg Leu Leu
1205 1210 1215
Lys Leu Leu Cys Val Pro Phe Gly Tyr Cys Ile Pro Arg Phe Lys
1220 1225 1230
Asn Leu Ser Ile Glu Ala Leu Phe Asp Arg Asn Glu Ala Ala Lys
1235 1240 1245
Phe Asp Lys Glu Lys Lys Lys Val Ser Gly Asn Ser Gly Ser Lys
1250 1255 1260
Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1265 1270 1275
Lys Val
1280
<210> 95
<211> 1266
<212> PRT
<213> Artificial Sequence
<220>
<223> CURE-X3
<400> 95
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val Gly Ser Ile Glu Lys Lys Lys Ser Phe Ala Lys Gly Met Gly
20 25 30
Val Lys Ser Thr Leu Val Ser Gly Ser Lys Val Tyr Met Thr Thr Phe
35 40 45
Ala Glu Gly Ser Asp Ala Arg Leu Glu Lys Ile Val Glu Gly Asp Ser
50 55 60
Ile Arg Ser Val Asn Glu Gly Glu Ala Phe Ser Ala Glu Met Ala Asp
65 70 75 80
Lys Asn Ala Gly Tyr Lys Ile Gly Asn Ala Lys Phe Ser His Pro Lys
85 90 95
Gly Tyr Ala Val Val Ala Asn Asn Pro Leu Tyr Thr Gly Pro Val Gln
100 105 110
Gln Asp Met Leu Gly Leu Lys Glu Thr Leu Glu Lys Arg Tyr Phe Gly
115 120 125
Glu Ser Ala Asp Gly Asn Asp Asn Ile Cys Ile Gln Val Ile His Asn
130 135 140
Ile Leu Asp Ile Glu Lys Ile Leu Ala Glu Tyr Ile Thr Asn Ala Ala
145 150 155 160
Tyr Ala Val Asn Asn Ile Ser Gly Leu Asp Lys Asp Ile Ile Gly Phe
165 170 175
Gly Lys Phe Ser Thr Val Tyr Thr Tyr Asp Glu Phe Lys Asp Pro Glu
180 185 190
His His Arg Ala Ala Phe Asn Asn Asn Asp Lys Leu Ile Asn Ala Ile
195 200 205
Lys Ala Gln Tyr Asp Glu Phe Asp Asn Phe Leu Asp Asn Pro Arg Leu
210 215 220
Gly Tyr Phe Gly Gln Ala Phe Phe Ser Lys Glu Gly Arg Asn Tyr Ile
225 230 235 240
Ile Asn Tyr Gly Asn Glu Cys Tyr Asp Ile Leu Ala Leu Leu Ser Gly
245 250 255
Leu Ala His Trp Val Val Ala Asn Asn Glu Glu Glu Ser Arg Ile Ser
260 265 270
Arg Thr Trp Leu Tyr Asn Leu Asp Lys Asn Leu Asp Asn Glu Tyr Ile
275 280 285
Ser Thr Leu Asn Tyr Leu Tyr Asp Arg Ile Thr Asn Glu Leu Thr Asn
290 295 300
Ser Phe Ser Lys Asn Ser Ala Ala Asn Val Asn Tyr Ile Ala Glu Thr
305 310 315 320
Leu Gly Ile Asn Pro Ala Glu Phe Ala Glu Gln Tyr Phe Arg Phe Ser
325 330 335
Ile Met Lys Glu Gln Lys Asn Leu Gly Phe Asn Ile Thr Lys Leu Arg
340 345 350
Glu Val Met Leu Asp Arg Lys Asp Met Ser Glu Ile Arg Lys Asn His
355 360 365
Lys Val Phe Asp Ser Ile Arg Thr Lys Val Tyr Thr Met Met Asp Phe
370 375 380
Val Ile Tyr Arg Tyr Tyr Ile Glu Glu Asp Ala Lys Val Ala Ala Ala
385 390 395 400
Asn Lys Ser Leu Pro Asp Asn Glu Lys Ser Leu Ser Glu Lys Asp Ile
405 410 415
Phe Val Ile Asn Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys Asp Ala
420 425 430
Leu Tyr Tyr Asp Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu Asn Ile
435 440 445
Met His Asn Ile Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu Tyr Lys
450 455 460
Lys Lys Asp Ala Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly Arg Asp
465 470 475 480
Val Ser Ala Phe Ser Lys Leu Met Tyr Ala Leu Thr Met Phe Leu Asp
485 490 495
Gly Lys Glu Ile Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe Asp
500 505 510
Asn Ile Gln Ser Phe Leu Lys Val Met Pro Leu Ile Gly Val Asn Ala
515 520 525
Lys Phe Val Glu Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys Ile Ala
530 535 540
Asp Glu Leu Arg Leu Ile Lys Ser Phe Ala Arg Met Gly Glu Pro Ile
545 550 555 560
Ala Asp Ala Arg Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile Leu Gly
565 570 575
Thr Asn Gly Ser Asn Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu
580 585 590
Ser Pro Lys Lys Lys Arg Lys Val Gly Gly Ser Gly Gly Ser Gly Gly
595 600 605
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ala Ala Ala Gly Ser Met
610 615 620
Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile
625 630 635 640
Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu
645 650 655
Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp
660 665 670
Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly
675 680 685
Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser
690 695 700
Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser
705 710 715 720
Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe
725 730 735
Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg Ile
740 745 750
Tyr Asp Asp Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp
755 760 765
Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His Cys
770 775 780
Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp Asp
785 790 795 800
Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile
805 810 815
Leu Gln Asn Gln Gly Asn Ala Ala Ala Gly Ser Gly Gly Ser Gly Gly
820 825 830
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Arg Thr
835 840 845
Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val Gly
850 855 860
Pro Gly Ser Arg Asn Phe Ile Ile Asn Asn Val Ile Ser Asn Lys Arg
865 870 875 880
Phe His Tyr Leu Ile Arg Tyr Gly Asp Pro Ala His Leu His Glu Ile
885 890 895
Ala Lys Asn Glu Ala Val Val Lys Phe Val Leu Gly Arg Ile Ala Asp
900 905 910
Ile Gln Lys Lys Gln Gly Gln Asn Gly Lys Asn Gln Ile Asp Arg Tyr
915 920 925
Tyr Glu Thr Cys Ile Gly Lys Asp Lys Gly Lys Ser Val Ser Glu Lys
930 935 940
Val Asp Ala Leu Thr Lys Ile Ile Thr Gly Met Asn Tyr Asp Gln Phe
945 950 955 960
Asp Lys Lys Arg Ser Val Ile Glu Asp Thr Gly Arg Glu Asn Ala Glu
965 970 975
Arg Glu Lys Phe Lys Lys Ile Ile Ser Leu Tyr Leu Thr Val Ile Tyr
980 985 990
His Ile Leu Lys Asn Ile Val Asn Ile Asn Ala Arg Tyr Val Ile Gly
995 1000 1005
Phe His Cys Val Glu Arg Asp Ala Gln Leu Tyr Lys Glu Lys Gly
1010 1015 1020
Tyr Asp Ile Asn Leu Lys Lys Leu Glu Glu Lys Gly Phe Ser Ser
1025 1030 1035
Val Thr Lys Leu Cys Ala Gly Ile Asp Glu Thr Ala Pro Asp Lys
1040 1045 1050
Arg Lys Asp Val Glu Lys Glu Met Ala Glu Arg Ala Lys Glu Ser
1055 1060 1065
Ile Asp Ser Leu Glu Ser Ala Asn Pro Lys Leu Tyr Ala Asn Tyr
1070 1075 1080
Ile Lys Tyr Ser Asp Glu Lys Lys Ala Glu Glu Phe Thr Arg Gln
1085 1090 1095
Ile Asn Arg Glu Lys Ala Lys Thr Ala Leu Asn Ala Tyr Leu Arg
1100 1105 1110
Asn Thr Lys Trp Asn Val Ile Ile Arg Glu Asp Leu Leu Arg Ile
1115 1120 1125
Asp Asn Lys Thr Cys Thr Leu Phe Ala Asn Lys Ala Val Ala Leu
1130 1135 1140
Glu Val Ala Arg Tyr Val His Ala Tyr Ile Asn Asp Ile Ala Glu
1145 1150 1155
Val Asn Ser Tyr Phe Gln Leu Tyr His Tyr Ile Met Gln Arg Ile
1160 1165 1170
Ile Met Asn Glu Arg Tyr Glu Lys Ser Ser Gly Lys Val Ser Glu
1175 1180 1185
Tyr Phe Asp Ala Val Asn Asp Glu Lys Lys Tyr Asn Asp Arg Leu
1190 1195 1200
Leu Lys Leu Leu Cys Val Pro Phe Gly Tyr Cys Ile Pro Arg Phe
1205 1210 1215
Lys Asn Leu Ser Ile Glu Ala Leu Phe Asp Arg Asn Glu Ala Ala
1220 1225 1230
Lys Phe Asp Lys Glu Lys Lys Lys Val Ser Gly Asn Ser Gly Ser
1235 1240 1245
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1250 1255 1260
Arg Lys Val
1265
<210> 96
<211> 1229
<212> PRT
<213> Artificial Sequence
<220>
<223> CURE-X4
<400> 96
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val Ala Ala Ala Gly Ser Met Glu Ala Ser Pro Ala Ser Gly Pro
20 25 30
Arg His Leu Met Asp Pro His Ile Phe Thr Ser Asn Phe Asn Asn Gly
35 40 45
Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp
50 55 60
Asn Gly Thr Ser Val Lys Met Asp Gln His Arg Gly Phe Leu His Asn
65 70 75 80
Gln Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu
85 90 95
Arg Phe Leu Asp Leu Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile
100 105 110
Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly
115 120 125
Cys Ala Gly Glu Val Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg
130 135 140
Leu Arg Ile Phe Ala Ala Arg Ile Tyr Asp Asp Asp Pro Leu Tyr Lys
145 150 155 160
Glu Ala Leu Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met
165 170 175
Thr Tyr Asp Glu Phe Lys His Cys Trp Asp Thr Phe Val Asp His Gln
180 185 190
Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala
195 200 205
Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn Ala Ala
210 215 220
Ala Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
225 230 235 240
Gly Gly Ser Ile Glu Lys Lys Lys Ser Phe Ala Lys Gly Met Gly Val
245 250 255
Lys Ser Thr Leu Val Ser Gly Ser Lys Val Tyr Met Thr Thr Phe Ala
260 265 270
Glu Gly Ser Asp Ala Arg Leu Glu Lys Ile Val Glu Gly Asp Ser Ile
275 280 285
Arg Ser Val Asn Glu Gly Glu Ala Phe Ser Ala Glu Met Ala Asp Lys
290 295 300
Asn Ala Gly Tyr Lys Ile Gly Asn Ala Lys Phe Ser His Pro Lys Gly
305 310 315 320
Tyr Ala Val Val Ala Asn Asn Pro Leu Tyr Thr Gly Pro Val Gln Gln
325 330 335
Asp Met Leu Gly Leu Lys Glu Thr Leu Glu Lys Arg Tyr Phe Gly Glu
340 345 350
Ser Ala Asp Gly Asn Asp Asn Ile Cys Ile Gln Val Ile His Asn Ile
355 360 365
Leu Asp Ile Glu Lys Ile Leu Ala Glu Tyr Ile Thr Asn Ala Ala Tyr
370 375 380
Ala Val Asn Asn Ile Ser Gly Leu Asp Lys Asp Ile Ile Gly Phe Gly
385 390 395 400
Lys Phe Ser Thr Val Tyr Thr Tyr Asp Glu Phe Lys Asp Pro Glu His
405 410 415
His Arg Ala Ala Phe Asn Asn Asn Asp Lys Leu Ile Asn Ala Ile Lys
420 425 430
Ala Gln Tyr Asp Glu Phe Asp Asn Phe Leu Asp Asn Pro Arg Leu Gly
435 440 445
Tyr Phe Gly Gln Ala Phe Phe Ser Lys Glu Gly Arg Asn Tyr Ile Ile
450 455 460
Asn Tyr Gly Asn Glu Cys Tyr Asp Ile Leu Ala Leu Leu Ser Gly Leu
465 470 475 480
Ala His Trp Val Val Ala Asn Asn Glu Glu Glu Ser Arg Ile Ser Arg
485 490 495
Thr Trp Leu Tyr Asn Leu Asp Lys Asn Leu Asp Asn Glu Tyr Ile Ser
500 505 510
Thr Leu Asn Tyr Leu Tyr Asp Arg Ile Thr Asn Glu Leu Thr Asn Ser
515 520 525
Phe Ser Lys Asn Ser Ala Ala Asn Val Asn Tyr Ile Ala Glu Thr Leu
530 535 540
Gly Ile Asn Pro Ala Glu Phe Ala Glu Gln Tyr Phe Arg Phe Ser Ile
545 550 555 560
Met Lys Glu Gln Lys Asn Leu Gly Phe Asn Ile Thr Lys Leu Arg Glu
565 570 575
Val Met Leu Asp Arg Lys Asp Met Ser Glu Ile Arg Lys Asn His Lys
580 585 590
Val Phe Asp Ser Ile Arg Thr Lys Val Tyr Thr Met Met Asp Phe Val
595 600 605
Ile Tyr Arg Tyr Tyr Ile Glu Glu Asp Ala Lys Val Ala Ala Ala Asn
610 615 620
Lys Ser Leu Pro Asp Asn Glu Lys Ser Leu Ser Glu Lys Asp Ile Phe
625 630 635 640
Val Ile Asn Leu Arg Gly Ser Phe Asn Asp Asp Gln Lys Asp Ala Leu
645 650 655
Tyr Tyr Asp Glu Ala Asn Arg Ile Trp Arg Lys Leu Glu Asn Ile Met
660 665 670
His Asn Ile Lys Glu Phe Arg Gly Asn Lys Thr Arg Glu Tyr Lys Lys
675 680 685
Lys Asp Ala Pro Arg Leu Pro Arg Ile Leu Pro Ala Gly Arg Asp Val
690 695 700
Ser Ala Phe Ser Lys Leu Met Tyr Ala Leu Thr Met Phe Leu Asp Gly
705 710 715 720
Lys Glu Ile Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe Asp Asn
725 730 735
Ile Gln Ser Phe Leu Lys Val Met Pro Leu Ile Gly Val Asn Ala Lys
740 745 750
Phe Val Glu Glu Tyr Ala Phe Phe Lys Asp Ser Ala Lys Ile Ala Asp
755 760 765
Glu Leu Arg Leu Ile Lys Ser Phe Ala Arg Met Gly Glu Pro Ile Ala
770 775 780
Asp Ala Arg Arg Ala Met Tyr Ile Asp Ala Ile Arg Ile Leu Gly Thr
785 790 795 800
Asn Leu Ser Tyr Asp Glu Leu Lys Ala Leu Ala Asp Thr Phe Ser Leu
805 810 815
Asp Glu Asn Gly Asn Lys Leu Lys Lys Gly Lys His Gly Met Arg Asn
820 825 830
Phe Ile Ile Asn Asn Val Ile Ser Asn Lys Arg Phe His Tyr Leu Ile
835 840 845
Arg Tyr Gly Asp Pro Ala His Leu His Glu Ile Ala Lys Asn Glu Ala
850 855 860
Val Val Lys Phe Val Leu Gly Arg Ile Ala Asp Ile Gln Lys Lys Gln
865 870 875 880
Gly Gln Asn Gly Lys Asn Gln Ile Asp Arg Tyr Tyr Glu Thr Cys Ile
885 890 895
Gly Lys Asp Lys Gly Lys Ser Val Ser Glu Lys Val Asp Ala Leu Thr
900 905 910
Lys Ile Ile Thr Gly Met Asn Tyr Asp Gln Phe Asp Lys Lys Arg Ser
915 920 925
Val Ile Glu Asp Thr Gly Arg Glu Asn Ala Glu Arg Glu Lys Phe Lys
930 935 940
Lys Ile Ile Ser Leu Tyr Leu Thr Val Ile Tyr His Ile Leu Lys Asn
945 950 955 960
Ile Val Asn Ile Asn Ala Arg Tyr Val Ile Gly Phe His Cys Val Glu
965 970 975
Arg Asp Ala Gln Leu Tyr Lys Glu Lys Gly Tyr Asp Ile Asn Leu Lys
980 985 990
Lys Leu Glu Glu Lys Gly Phe Ser Ser Val Thr Lys Leu Cys Ala Gly
995 1000 1005
Ile Asp Glu Thr Ala Pro Asp Lys Arg Lys Asp Val Glu Lys Glu
1010 1015 1020
Met Ala Glu Arg Ala Lys Glu Ser Ile Asp Ser Leu Glu Ser Ala
1025 1030 1035
Asn Pro Lys Leu Tyr Ala Asn Tyr Ile Lys Tyr Ser Asp Glu Lys
1040 1045 1050
Lys Ala Glu Glu Phe Thr Arg Gln Ile Asn Arg Glu Lys Ala Lys
1055 1060 1065
Thr Ala Leu Asn Ala Tyr Leu Arg Asn Thr Lys Trp Asn Val Ile
1070 1075 1080
Ile Arg Glu Asp Leu Leu Arg Ile Asp Asn Lys Thr Cys Thr Leu
1085 1090 1095
Phe Ala Asn Lys Ala Val Ala Leu Glu Val Ala Arg Tyr Val His
1100 1105 1110
Ala Tyr Ile Asn Asp Ile Ala Glu Val Asn Ser Tyr Phe Gln Leu
1115 1120 1125
Tyr His Tyr Ile Met Gln Arg Ile Ile Met Asn Glu Arg Tyr Glu
1130 1135 1140
Lys Ser Ser Gly Lys Val Ser Glu Tyr Phe Asp Ala Val Asn Asp
1145 1150 1155
Glu Lys Lys Tyr Asn Asp Arg Leu Leu Lys Leu Leu Cys Val Pro
1160 1165 1170
Phe Gly Tyr Cys Ile Pro Arg Phe Lys Asn Leu Ser Ile Glu Ala
1175 1180 1185
Leu Phe Asp Arg Asn Glu Ala Ala Lys Phe Asp Lys Glu Lys Lys
1190 1195 1200
Lys Val Ser Gly Asn Ser Gly Ser Lys Arg Thr Ala Asp Gly Ser
1205 1210 1215
Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val
1220 1225
<210> 97
<211> 1181
<212> DNA
<213> Artificial Sequence
<220>
<223> 以TC1316扩增出的第二dCas13Rx片段
<400> 97
atgcgcaact tcatcatcaa caacgtgatc agcaacaagc ggtttcacta cctgatcaga 60
tacggcgacc cagctcacct gcacgagatc gctaagaacg aggccgtggt gaagttcgtg 120
ctgggacgga tcgccgatat ccagaagaag cagggccaga acggaaagaa ccagatcgac 180
cgctactacg agacctgcat cggcaaggat aagggaaagt ccgtgtctga gaaggtggac 240
gctctgacca agatcatcac aggcatgaac tacgaccagt tcgataagaa gagatctgtg 300
atcgaggaca ccggaaggga gaacgccgag agagagaagt ttaagaagat catcagcctg 360
tacctgacag tgatctacca catcctgaag aacatcgtga acatcaacgc tagatacgtg 420
atcggcttcc actgcgtgga gcgcgatgcc cagctgtaca aggagaaggg atacgacatc 480
aacctgaaga agctggagga gaagggcttt agctccgtga ccaagctgtg cgctggaatc 540
gacgagacag cccccgacaa gaggaaggat gtggagaagg agatggccga gagagctaag 600
gagagcatcg actccctgga gtctgctaac cctaagctgt acgccaacta catcaagtac 660
tccgatgaga agaaggccga ggagttcacc aggcagatca acagagagaa ggccaagacc 720
gctctgaacg cctacctgag gaacacaaag tggaacgtga tcatccggga ggacctgctg 780
cgcatcgata acaagacctg tacactgttc gctaacaagg ctgtggccct ggaggtggct 840
cgctacgtgc acgcctacat caacgacatc gccgaggtga actcctactt tcagctgtac 900
cactacatca tgcagaggat catcatgaac gagagatacg agaagtctag cggcaaggtg 960
tctgagtact tcgacgccgt gaacgatgag aagaagtaca acgatagact gctgaagctg 1020
ctgtgcgtgc ctttcggata ctgtatccca cggtttaaga acctgagcat cgaggccctg 1080
ttcgaccgca acgaggctgc caagtttgat aaggagaaga agaaggtgag cggcaactcc 1140
ggttctaagc gcaccgccga tggatccgag ttcgaatccc c 1181
<210> 98
<211> 81
<212> DNA
<213> Artificial Sequence
<220>
<223> 以TC1316扩增出的第三bpNLS片段
<400> 98
ggatccaaaa gaactgcgga tggatctgag tttgagagtc caaaaaagaa gaggaaggtc 60
ggttctatgc gcaacttcat c 81
<210> 99
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> GS linker
<400> 99
ggsggsggsg gsggsggs 18
<210> 100
<211> 8174
<212> DNA
<213> Artificial Sequence
<220>
<223> TC1614:
CMV-BPNLS-dCasRx(1I-558G)-BPNLS-Apobec3A(Y132D)-BPNLS-dCasRx(587M
-966S)-BPNLS序列
<400> 100
aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 60
ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 120
aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga 180
tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 240
cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa 300
gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt 360
tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt 420
tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 480
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 540
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 600
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 660
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 720
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 780
gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 840
ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 900
tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 960
tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt 1020
atcgaaatta atacgactca ctatagggag acccaagctg gctagcgttt aaacttaagc 1080
ttgccaccat gaaacggact gctgacggca gcgaatttga gagccccaag aagaagagga 1140
aagtcggatc catcgagaag aagaagagct tcgccaaggg catgggagtg aagagcaccc 1200
tggtgtccgg ctctaaggtg tacatgacca catttgctga gggaagcgac gccaggctgg 1260
agaagatcgt ggagggcgat agcatcagat ccgtgaacga gggagaggct ttcagcgccg 1320
agatggctga caagaacgct ggctacaaga tcggaaacgc caagttttcc cacccaaagg 1380
gctacgccgt ggtggctaac aacccactgt acaccggacc agtgcagcag gacatgctgg 1440
gactgaagga gacactggag aagaggtact tcggcgagtc cgccgacgga aacgataaca 1500
tctgcatcca ggtcatccac aacatcctgg atatcgagaa gatcctggct gagtacatca 1560
caaacgccgc ttacgccgtg aacaacatct ccggcctgga caaggatatc atcggcttcg 1620
gaaagttttc taccgtgtac acatacgacg agttcaagga tccagagcac caccgggccg 1680
cttttaacaa caacgacaag ctgatcaacg ccatcaaggc tcagtacgac gagttcgata 1740
actttctgga taaccccagg ctgggctact tcggacaggc tttcttttct aaggagggca 1800
gaaactacat catcaactac ggaaacgagt gttacgacat cctggccctg ctgagcggac 1860
tggcccactg ggtggtggcc aacaacgagg aggagtctcg gatcagccgc acctggctgt 1920
acaacctgga caagaacctg gataacgagt acatctccac actgaactac ctgtacgaca 1980
ggatcaccaa cgagctgaca aacagcttct ccaagaactc tgccgctaac gtgaactaca 2040
tcgctgagac cctgggcatc aacccagctg agttcgctga gcagtacttc agattttcca 2100
tcatgaagga gcagaagaac ctgggcttca acatcacaaa gctgagagaa gtgatgctgg 2160
acagaaagga tatgtccgag atcaggaaga accacaaggt gttcgattct atcagaacca 2220
aggtgtacac aatgatggac tttgtgatct acaggtacta catcgaggag gatgccaagg 2280
tggccgctgc caacaagagc ctgcccgaca acgagaagtc tctgagcgag aaggatatct 2340
tcgtgatcaa cctgagaggc tcctttaacg acgatcagaa ggacgctctg tactacgatg 2400
aggccaacag gatctggaga aagctggaga acatcatgca caacatcaag gagttccggg 2460
gaaacaagac ccgcgagtac aagaagaagg acgctccaag gctgcctagg atcctgcctg 2520
ctggaaggga cgtgagcgcc ttcagcaagc tgatgtacgc cctgacaatg tttctggacg 2580
gaaaggagat caacgatctg ctgaccacac tgatcaacaa gttcgacaac atccagtctt 2640
ttctgaaagt gatgcctctg atcggcgtga acgctaagtt cgtggaggag tacgccttct 2700
ttaaggacag cgccaagatc gctgatgagc tgcggctgat caagtccttt gccaggatgg 2760
gagagccaat cgctgacgct aggagagcta tgtacatcga tgccatccgg atcctgggaa 2820
ccaacggtac cagcaagagg acagcagacg ggtccgaatt cgaatccccc aagaaaaaga 2880
ggaaggtggg tcccgggtct atggaagcca gcccagcatc cgggcccaga cacttgatgg 2940
atccacacat attcacttcc aactttaaca atggcattgg aaggcataag acctacctgt 3000
gctacgaagt ggagcgcctg gacaatggca cctcggtcaa gatggaccag cacaggggct 3060
ttctacacaa ccaggctaag aatcttctct gtggctttta cggccgccat gcggagctgc 3120
gcttcttgga cctggttcct tctttgcagt tggacccggc ccagatctac agggtcactt 3180
ggttcatctc ctggagcccc tgcttctcct ggggctgtgc cggggaagtg cgtgcgttcc 3240
ttcaggagaa cacacacgtg agactgcgta tcttcgctgc ccgcatctat gatgacgacc 3300
ccctatataa ggaggcactg caaatgctgc gggatgctgg ggcccaagtc tccatcatga 3360
cctacgatga atttaagcac tgctgggaca cctttgtgga ccaccaggga tgtcccttcc 3420
agccctggga tggactagat gagcacagcc aagccctgag tgggaggctg cgggccattc 3480
tccagaatca gggaaacgga tccaaaagaa ctgcggatgg atctgagttt gagagtccaa 3540
aaaagaagag gaaggtcggt tctatgcgca acttcatcat caacaacgtg atcagcaaca 3600
agcggtttca ctacctgatc agatacggcg acccagctca cctgcacgag atcgctaaga 3660
acgaggccgt ggtgaagttc gtgctgggac ggatcgccga tatccagaag aagcagggcc 3720
agaacggaaa gaaccagatc gaccgctact acgagacctg catcggcaag gataagggaa 3780
agtccgtgtc tgagaaggtg gacgctctga ccaagatcat cacaggcatg aactacgacc 3840
agttcgataa gaagagatct gtgatcgagg acaccggaag ggagaacgcc gagagagaga 3900
agtttaagaa gatcatcagc ctgtacctga cagtgatcta ccacatcctg aagaacatcg 3960
tgaacatcaa cgctagatac gtgatcggct tccactgcgt ggagcgcgat gcccagctgt 4020
acaaggagaa gggatacgac atcaacctga agaagctgga ggagaagggc tttagctccg 4080
tgaccaagct gtgcgctgga atcgacgaga cagcccccga caagaggaag gatgtggaga 4140
aggagatggc cgagagagct aaggagagca tcgactccct ggagtctgct aaccctaagc 4200
tgtacgccaa ctacatcaag tactccgatg agaagaaggc cgaggagttc accaggcaga 4260
tcaacagaga gaaggccaag accgctctga acgcctacct gaggaacaca aagtggaacg 4320
tgatcatccg ggaggacctg ctgcgcatcg ataacaagac ctgtacactg ttcgctaaca 4380
aggctgtggc cctggaggtg gctcgctacg tgcacgccta catcaacgac atcgccgagg 4440
tgaactccta ctttcagctg taccactaca tcatgcagag gatcatcatg aacgagagat 4500
acgagaagtc tagcggcaag gtgtctgagt acttcgacgc cgtgaacgat gagaagaagt 4560
acaacgatag actgctgaag ctgctgtgcg tgcctttcgg atactgtatc ccacggttta 4620
agaacctgag catcgaggcc ctgttcgacc gcaacgaggc tgccaagttt gataaggaga 4680
agaagaaggt gagcggcaac tccggttcta agcgcaccgc cgatggatcc gagttcgaat 4740
cccctaaaaa gaaaagaaag gtgggatccg cggccgctgg atccgctact aacttcagcc 4800
tgctgaagca ggctggagac gtggaggaga accctggacc tatggtgagc aagggcgagg 4860
aggataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg 4920
tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc 4980
agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt 5040
cccctcagtt catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact 5100
acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg 5160
gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg 5220
tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg 5280
gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca 5340
agcagaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca 5400
aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca 5460
cctcccacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact 5520
ccaccggcgg catggacgag ctgtacaagt aaggccgcga ctctagagtc gacctgcagg 5580
catgcaagct tgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc 5640
ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg 5700
ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc 5760
tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg 5820
acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg 5880
ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga 5940
caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct 6000
ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg 6060
tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc 6120
ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc 6180
cgcatcgata ccgtcgacct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc 6240
ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa 6300
tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 6360
gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg agcttcctcg 6420
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 6480
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 6540
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 6600
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6660
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6720
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6780
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6840
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6900
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6960
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 7020
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 7080
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtttttt tgtttgcaag 7140
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 7200
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 7260
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 7320
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 7380
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 7440
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 7500
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 7560
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 7620
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 7680
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 7740
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 7800
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 7860
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 7920
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 7980
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 8040
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 8100
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 8160
gcaaaaaagg gaat 8174
<210> 101
<211> 315
<212> DNA
<213> Artificial Sequence
<220>
<223> 荧光报告系统质粒-第二质粒
<400> 101
aaggttagcg cactgaagga aaaagtttca gcactgaaag agaaggtgag tgccctcaaa 60
gagaaagtct ccgctctcaa ggaaaaagtc tcagccctta aagagggagg atctggcggc 120
agggaccaca tggtcctgca cgagtacgtc aatgcagcag gaatcaccgg atcctcgaat 180
attacgaccc tcgagggcgg cagcggcggc gaagtaagtg ctcttgaaaa agaggtgtct 240
gcccttgaaa aggaagtctc agcactggaa aaggaggtat ccgcacttga gaaggaggta 300
agtgccctgg agaag 315

Claims (12)

1.一种融合蛋白，其特征在于，其包括APOBEC 3A片段和失活的dCas13片段，所述失活的dCas13片段包括dCas13Rx片段和/或dPspCas13b片段。
2.如权利要求1所述的融合蛋白，其特征在于，所述APOBEC 3A片段的氨基酸序列如SEQID NO:85所示；或者所述APOBEC 3A片段包含突变Y132D和/或H29K，其氨基酸序列优选如SEQ ID NO:2或SEQ ID NO:90所示；
和/或，所述dPspCas13b片段的氨基酸序列如SEQ ID NO:1所示；
和/或，所述dCas13Rx片段的氨基酸序列如SEQ ID NO:91所示；
和/或，所述dPspCas13b片段位于所述APOBEC 3A片段的N端；
和/或，所述dCas13Rx片段位于所述APOBEC 3A片段的C端或插入所述APOBEC3A片段中间；例如将所述dCas13Rx片段的loop3位点、所述dCas13Rx片段的第178-192位、或所述dCas13Rx片段的第377-391位替换为所述APOBEC 3A片段；所述APOBEC 3A蛋白与所述dCas13Rx优选通过接头相连，所述接头的序列优选如SEQ ID NO:99所示；当将所述dCas13Rx片段的loop3位点替换为所述APOBEC 3A片段时，所述dCas13Rx片段被分为第一dCas13Rx片段和第二dCas13Rx片段，所述第一dCas13Rx片段的氨基酸序列优选如SEQ IDNO:3所示，所述第二dCas13Rx片段的氨基酸序列优选如SEQ ID NO:4所示；
和/或，所述的融合蛋白还包括一个或多个NLS或其突变，例如包括两个NLS或其突变；所述NLS例如为SV40 NLS和/或bpNLS；所述NLS或其突变的氨基酸序列优选如SEQ ID NO:5或SEQ ID NO:6所示；所述NLS优选位于所述融合蛋白的N端和/或C端、和/或插入所述融合蛋白的中间；
和/或，所述的融合蛋白还包括NES片段，例如HIV NES片段；
和/或，所述的融合蛋白用于碱基编辑，优选用于C到U的碱基编辑；
较佳地：
所述融合蛋白自N端至C端依次包括NLS或其突变、dPspCas13b片段和APOBEC3A片段；
或者，所述融合蛋白自N端至C端依次包括NLS、dPspCas13b片段、APOBEC 3A片段、和NLS；
或者，所述融合蛋白自N端至C端依次包括NLS或其突变、dPspCas13b片段、HIV NES片段和APOBEC 3A片段；
或者，所述融合蛋白自N端至C端依次包括NLS、dPspCas13b片段、HIV NES片段、APOBEC3A片段和NLS；
或者，当所述dCas13Rx片段插入所述APOBEC 3A片段中间时，所述dCas13Rx片段被分为第一dCas13Rx片段和第二dCas13Rx片段，所述融合蛋白自N端至C端依次包括NLS、第一dCas13Rx片段、NLS、APOBEC 3A片段、NLS、第二dCas13Rx片段、和NLS；
或者，当所述dCas13Rx片段插入所述APOBEC 3A片段中间时，所述dCas13Rx片段被分为第一dCas13Rx片段和第二dCas13Rx片段，所述融合蛋白自N端至C端依次包括NLS、第一dCas13Rx片段、NLS、接头、APOBEC 3A片段、接头、NLS、第二dCas13Rx片段、和NLS；
或者，所述融合蛋白自N端至C端依次包括NLS、APOBEC 3A片段、接头、dCas13Rx片段和NLS；
更佳地：
所述融合蛋白的氨基酸序列包括如SEQ ID NO:7-10、93-96任一项所示的氨基酸序列；编码所述融合蛋白的核苷酸序列优选如SEQ ID NO:17、20、25、28、31、34、48任一项所示。
3.一种分离的多核苷酸，其编码如权利要求1或2所述的融合蛋白；
较佳地，其核苷酸序列如SEQ ID NO:17、20、25、28、31、34、48任一项所示。
4.一种构建体，其含有如权利要求3所述的分离的多核苷酸。
5.一种表达系统，所述表达系统含有如权利要求4所述的构建体或基因组中整合有如权利要求3所述的多核苷酸；
较佳地，所述表达系统的宿主细胞选自真核细胞或原核细胞，优选自人细胞，更优选自人源胚胎肾细胞例如293T。
6.一种融合蛋白的制备方法，其包括以下步骤：
(1)获得如权利要求5所述的表达系统；
(2)筛选所述表达系统，表达并纯化所述融合蛋白。
7.一种碱基编辑器，其包括如权利要求1或2所述的融合蛋白、如权利要求3所述的多核苷酸、如权利要求4所述的构建体、如权利要求5所述的表达系统；
较佳地：
所述碱基编辑器还包括指导RNA；所述指导RNA优选为包括28-36个碱基，优先为32个碱基；所述指导RNA优选其第5位不为C；所述指导RNA的序列更优选如SEQ ID NO:92所示；
和/或，碱基编辑器用于编辑核酸序列的编辑框，所述编辑框优选包括6-20个碱基，例如为8、10、12、14或18个碱基；
和/或，所述碱基编辑器用于真核生物的碱基编辑；
和/或，所述碱基编辑器用于体外的碱基编辑；
和/或，所述碱基编辑器为C到U的碱基编辑器；
和/或，所述碱基编辑器中使用的荧光报告系统包括第一质粒和第二质粒，所述第一质粒的序列如SEQ ID NO:89所示，所述第二质粒的序列如SEQ ID NO:101所示。
8.一种试剂盒，其包括如权利要求1或2所述的融合蛋白、如权利要求3所述的多核苷酸、如权利要求4所述的构建体、如权利要求5所述的表达系统；
较佳地：
所述试剂盒还包括指导RNA；所述指导RNA优选为包括28-36个碱基，优先为32个碱基；所述指导RNA优选其第5位不为C；所述指导RNA的序列更优选如SEQ ID NO:92所示；
和/或，所述试剂盒用于编辑核酸序列的编辑框，所述编辑框优选包括6-20个碱基，例如为8、10、12、14或18个碱基；
和/或，所述试剂盒用于真核生物的碱基编辑；
和/或，所述试剂盒用于体外的碱基编辑；
和/或，所述试剂盒用于将C编辑为U；
和/或，所述碱基编辑器中使用的荧光报告系统包括第一质粒和第二质粒，所述第一质粒的序列如SEQ ID NO:89所示，所述第二质粒的序列如SEQ ID NO:101所示。
9.如权利要求1或2所述的融合蛋白、如权利要求3所述的分离的多核苷酸、如权利要求4所述的构建体、如权利要求5所述的表达系统、如权利要求7所述的碱基编辑器、或如权利要求8所述的试剂盒在碱基编辑中的用途；
较佳地：
所述用途为在真核生物的碱基编辑中的用途；
和/或，所述用途为在体外的碱基编辑中的用途；
和/或，所述用途为在将C编辑为U的碱基编辑器中的用途。
10.一种碱基编辑的方法，其特征在于，所述方法包括通过如权利要求1或2所述的融合蛋白、如权利要求3所述的多核苷酸、如权利要求4所述的构建体、如权利要求5所述的表达系统、如权利要求8所述的碱基编辑器、或如权利要求9所述的试剂盒进行碱基编辑的步骤；
较佳地：
所述方法包括使待碱基编辑的样品与指导RNA与所述的融合蛋白、所述的多核苷酸、所述的构建体、所述的表达系统、所述的碱基编辑器、或所述的试剂盒接触以进行碱基编辑；
和/或，所述碱基编辑为体外碱基编辑；
和/或，所述碱基编辑为真核生物的碱基编辑；
和/或，所述碱基编辑为将C编辑为U的碱基编辑。
11.一种荧光报告系统，其包括第一质粒和第二质粒，所述第一质粒的序列如SEQ IDNO:89所示，所述第二质粒的序列如SEQ ID NO:101所示。
12.如权利要求11所述的荧光报告系统在检测如权利要求1或2所述的融合蛋白的编辑效率中的应用。
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