本發(fā)明涉及基因組編輯領(lǐng)域，具體涉及一種增強的crispr堿基編輯系統(tǒng)實現(xiàn)高效率和高精度的堿基水平基因組編輯方法及其應(yīng)用。
背景技術(shù)：
：基因組編輯技術(shù)指利用可設(shè)計的核酸酶(分子剪刀)通過堿基插入、缺失或置換等方式，對生物體基因組dna特定片段進行改造從而達到對目標基因進行編輯的一種基因工程技術(shù)。利用基因組編輯技術(shù)對細胞進行遺傳學(xué)操縱，可廣泛的應(yīng)用于生命科學(xué)基礎(chǔ)研究、生物技術(shù)開發(fā)、農(nóng)業(yè)技術(shù)開發(fā)以及醫(yī)藥研發(fā)領(lǐng)域。例如：直接在體內(nèi)校正引起遺傳疾病的基因突變，將能夠從根本上治療遺傳疾?。粚r(nóng)作物進行精確的基因工程改造，使其產(chǎn)量提高或能夠抵抗環(huán)境污染或病原體的感染；對微生物基因組進行精準改造，從而促進可再生生物能源的開發(fā)等。crispr/cas(clusteredregularlyinterspacedshortpalindromicrepeats/crispr-associatedprotein)基因組編輯系統(tǒng)自問世以來，就有著其它基因組編輯技術(shù)無可比擬的優(yōu)勢，被認為能夠在活細胞中廣泛使用，并且是迄今為止最有效、最便捷的基因組編輯系統(tǒng)。cas9核酸酶利用引導(dǎo)rna(guiderna，grna)可在多種細胞的基因組特定靶點定位，對其進行切割從而產(chǎn)生dna雙鏈斷裂(doublestrandbreaks，dsbs)，然后利用細胞內(nèi)源的dna修復(fù)機制來實現(xiàn)編輯。根據(jù)不同dna修復(fù)通路的激活，基因組編輯將會導(dǎo)致基因的失活或者突變的校正。通常來說，有兩種主要的修復(fù)機制會被dsb所激活，一種是非同源末端連接(non-homologousendjoining，nhej)，另一種是同源性介導(dǎo)修復(fù)(homology-directedrepair，hdr)。做為dna雙鏈斷裂最主要的修復(fù)通路，nhej在修復(fù)過程中能在dsb附近的基因組位點引入隨機性堿基插入或缺失，從而導(dǎo)致基因的失活。與nhej相反，當hdr被激活時能夠以外源性供體dna作為模版，利用同源重組機制將外源性供體dna的序列替換靶點基因組dna的序列，從而完成基因突變的校正。但實際上，由于同源重組機制本身的局限性，hdr介導(dǎo)的基因校正效率一直很低(通常小于5％)。因此，極大限制了crispr/cas基因組編輯工具從科研向應(yīng)用的轉(zhuǎn)化，尤其是在精準基因治療方面的應(yīng)用，這也是基因編輯領(lǐng)域的一大難題。為了提高基因突變的校正效率，堿基編輯器(baseeditor，be)在近期內(nèi)應(yīng)運而生。這種堿基編輯器將crispr/cas系統(tǒng)和apobec胞嘧啶脫氨酶家族成員整合在一起，可執(zhí)行將胞嘧啶(cytosine，c)編輯為胸腺嘧啶(thymine，t)的功能。為了最大程度地提升c-t堿基編輯的效率，研究人員嘗試了將完全喪失酶活性的dcas9或者具有切刻酶活性的ncas9與apobec整合，從而產(chǎn)生了堿基編輯器2(be2)和堿基編輯器3(be3)。相對于含有dcas9的be2，含有ncas9的be3雖然在靶點序列有著更高的c-t編輯效率，但be3在靶點序列由crispr系統(tǒng)本身所引起的堿基插入或缺失(insertion/deletion，indel)率卻更高，反而降低了堿基編輯的功效(c-t堿基編輯率與堿基插入或缺失率的比值，c-teditingrate/indelrate)及精確度。因此，創(chuàng)造具有更高編輯功效的增強型堿基編輯系統(tǒng)，有助于實現(xiàn)高精度的堿基編輯，能極大地擴充我們對于堿基編輯器的應(yīng)用，特別是在醫(yī)療領(lǐng)域進行精準基因治療方面。技術(shù)實現(xiàn)要素：本發(fā)明的目的在于克服現(xiàn)有技術(shù)堿基編輯功效低的缺陷，提供一種堿基編輯系統(tǒng)及方法，其利用共表達be3與尿嘧啶糖苷酶抑制劑(uracildnaglycosylaseinhibitor，ugi)的方法提高堿基編輯器的功效，實現(xiàn)高精度的堿基編輯，從而可以更為有效的在各物種基因組中進行堿基編輯。為了達到上述目的，本發(fā)明提供了以下技術(shù)方案：一種堿基編輯系統(tǒng)，其特征在于，包括ugi表達載體或其轉(zhuǎn)錄產(chǎn)物和be3表達載體或其轉(zhuǎn)錄產(chǎn)物，或包括ugi和be3共表達載體或其轉(zhuǎn)錄產(chǎn)物。優(yōu)選地，所述的ugi和be3共表達載體為增強型crispr堿基編輯器ebea表達載體，其序列包括seqidno：22。優(yōu)選地，所述的ugi和be3共表達載體為增強型crispr堿基編輯器ebeb表達載體，其序列包括seqidno：23。優(yōu)選地，所述的ugi表達載體的序列包括seqidno：20。優(yōu)選地，所述的be3表達載體的序列包括seqidno：21。優(yōu)選地，所述的堿基編輯系統(tǒng)還包括針對某物種的基因組靶點的sgrna表達載體。優(yōu)選地，所述的堿基編輯系統(tǒng)還包括基因轉(zhuǎn)導(dǎo)試劑。優(yōu)選地，所述的堿基編輯系統(tǒng)還包括真核細胞。優(yōu)選地，所述的ugi表達載體、be3表達載體以及ugi和be3共表達載體包括但不限于質(zhì)粒載體。優(yōu)選地，所述的轉(zhuǎn)錄產(chǎn)物包括但不限于體外轉(zhuǎn)錄出的rna。本發(fā)明還提供了上述的堿基編輯系統(tǒng)的構(gòu)建方法，其特征在于，包括擴增ugi基因，并連入真核表達載體中，得到ugi表達載體；或利用pcr方法擴增t2a-ugi編碼序列，并同框連入be3編碼區(qū)的下游，得到增強型crispr堿基編輯器ebea表達載體；或人工合成3×t2a-ugi編碼序列，pcr擴增后同框連入be3編碼區(qū)的下游，得到到增強型crispr堿基編輯器ebeb表達載體。本發(fā)明還提供了上述的堿基編輯系統(tǒng)的應(yīng)用方法，其特征在于，包括構(gòu)建針對某物種的基因組dna靶點的sgrna；在真核細胞中共引入sgrna，be3表達載體或其轉(zhuǎn)錄產(chǎn)物以及ugi表達載體或其轉(zhuǎn)錄產(chǎn)物，或者，在真核細胞中共引入sgrna以及ugi和be3共表達載體或其轉(zhuǎn)錄產(chǎn)物，使其在靶點基因組dna發(fā)生c-t堿基編輯。優(yōu)選地，所述的堿基編輯系統(tǒng)的應(yīng)用方法還包括：對處理后的真核細胞基因組dna進行抽提，然后利用針對靶點的特異性引物進行pcr擴增，利用基因組dna的pcr產(chǎn)物構(gòu)建高通量二代dna測序文庫，并進行高通量二代dna測序。優(yōu)選地，所述的某物種的基因組dna靶點包括但不限于人的fancf和rnf2靶點中的至少一個。優(yōu)選地，所述的sgrna的序列為seqidno：24或seqidno：25。本發(fā)明提供了一種共表達be3與ugi的方法，首先通過構(gòu)建ugi單獨表達載體載體，并與be3表達載體和sgrna表達載體共轉(zhuǎn)染真核生物細胞，使其在靶點基因組dna發(fā)生c-t堿基編輯，從而提高了be3的功效。為進一步優(yōu)化實驗，本發(fā)明在be3編碼框的下游利用t2a自剪切肽段同框連接1×或者3×ugi基因，從而得到增強型crispr堿基編輯器ebea和ebeb表達載體，并將其與sgrna表達載體共轉(zhuǎn)染真核細胞，從而實現(xiàn)了高功效高精度的堿基編輯。本發(fā)明以人基因組dna靶點fancf(實施例1)和rnf2(實施例2)位點為例，實施了高功效高精度的c-t堿基編輯。與現(xiàn)有技術(shù)相比，本發(fā)明的有益效果是：本發(fā)明在國際上首次通過增加crispr堿基編輯器產(chǎn)生的c-t編輯率并降低crispr系統(tǒng)本身引起的堿基插入或缺失率，從而增強了crispr堿基編輯器的功效，為crispr堿基編輯器在各物種的基因組中實施更為準確和安全的堿基編輯提供了新的方法和思路。本發(fā)明所述的單/雙載體共表達crispr堿基編輯器與ugi的方法，可以在各種真核生物的基因組中實施高功效高精度的c-t堿基編輯。本發(fā)明所述的單/雙載體共表達crispr堿基編輯器和ugi的方法，可以提高多種crispr堿基編輯器的c-t堿基編輯率，并降低crispr系統(tǒng)本身引起的堿基插入或缺失率，從而增強多種crispr堿基編輯器的功效。附圖說明圖1a：實施例1雙載體共表達be3與ugi的方法示意圖圖1b：實施例1雙載體共表達be3與ugi的方法提高了堿基編輯器的功效。c6、c7、c8、c11代表其靶點序列中的第6、7、8、11位的胞嘧啶。圖1c：實施例1雙載體共表達be3與ugi的方法提高了堿基編輯器的c-t編輯率。c6、c7、c8、c11代表其靶點序列中的第6、7、8、11位的胞嘧啶。圖1d：實施例1雙載體共表達be3與ugi的方法抑制了crispr系統(tǒng)本身引起的堿基插入或缺失率。圖1e：實施例1單載體共表達be3與ugi的方法示意圖圖1f：實施例1單載體共表達be3與ugi的方法提高了堿基編輯器的功效。c6、c7、c8、c11代表其靶點序列中的第6、7、8、11位的胞嘧啶。圖1g：實施例1單載體共表達be3與ugi的方法提高了堿基編輯器的c-t編輯率。c6、c7、c8、c11代表其靶點序列中的第6、7、8、11位的胞嘧啶。圖1h：實施例1單載體共表達be3與ugi的方法抑制了crispr系統(tǒng)本身引起的堿基插入或缺失率。圖2a：實施例2雙載體共表達be3與ugi的方法示意圖圖2b：實施例2雙載體共表達be3與ugi的方法提高了堿基編輯器的功效。c3、c6代表其靶點序列中的第3、6位的胞嘧啶。圖2c：實施例2雙載體共表達be3與ugi的方法提高了堿基編輯器的c-t編輯率。c3、c6代表其靶點序列中的第3、6位的胞嘧啶。圖2d：實施例2雙載體共表達be3與ugi的方法抑制了crispr系統(tǒng)本身引起的堿基插入或缺失率。圖2e：實施例2單載體共表達be3與ugi的方法示意圖圖2f：實施例2單載體共表達be3與ugi的方法提高了堿基編輯器的功效。c3、c6代表其靶點序列中的第3、6位的胞嘧啶。圖2g：實施例2單載體共表達be3與ugi的方法提高了堿基編輯器的c-t編輯率。c3、c6代表其靶點序列中的第3、6位的胞嘧啶。圖2h：實施例2單載體共表達be3與ugi的方法抑制了crispr系統(tǒng)本身引起的堿基插入或缺失率。具體實施方式本發(fā)明的共表達be3與ugi的方法，首先通過構(gòu)建ugi單獨表達載體載體，并與be3表達載體和sgrna表達載體共轉(zhuǎn)染真核生物細胞，從而提高了be3的功效。為進一步優(yōu)化實驗，本發(fā)明在be3編碼框的下游利用t2a自剪切肽段同框連接1×或者3×ugi基因，從而得到增強型crispr堿基編輯器ebea和ebeb表達載體，并將其與sgrna表達載體共轉(zhuǎn)染真核細胞，從而實現(xiàn)了高功效高精度的堿基編輯。本發(fā)明以人基因組dna靶點fancf(實施例1)和rnf2(實施例2)位點為例，實施了高功效高精度的c-t堿基編輯。具體的，實驗方法包括下列步驟：1)構(gòu)建表達載體。將ugi基因從be3表達載體中利用pcr擴增出來，并連入真核表達載體pcdna3中，得到ugi單獨表達載體載體。利用pcr方法擴增t2a-ugi編碼序列，并同框連入be3編碼區(qū)的下游，得到到增強型crispr堿基編輯器ebea表達載體。人工合成3×t2a-ugi編碼序列，pcr擴增后同框連入be3編碼區(qū)的下游，得到到增強型crispr堿基編輯器ebeb表達載體。構(gòu)建針對人基因組dna靶點fancf和rnf2的sgrna。2)轉(zhuǎn)染真核細胞。在真核細胞中共轉(zhuǎn)染sgrna，be3表達載體以及低、中、高不同濃度的ugi表達載體。在真核細胞中共轉(zhuǎn)染sgrna和ebea、ebeb表達載體。3)基因組dna抽提擴增。抽提轉(zhuǎn)染后的真核細胞基因組dna，然后利用針對靶點fancf和rnf2的特異性引物進行pcr擴增。4)基因組dnapcr產(chǎn)物的高通量二代dna測序。利用基因組dna的pcr產(chǎn)物構(gòu)建高通量二代dna測序文庫，并進行高通量二代dna測序。本發(fā)明所述的單/雙載體共表達crispr堿基編輯器與ugi的方法，可以在各種真核生物的基因組中實施高功效高精度的c-t堿基編輯。本發(fā)明所述的單/雙載體共表達crispr堿基編輯器和ugi的方法，可以提高多種crispr堿基編輯器的c-t堿基編輯率，并降低crispr系統(tǒng)本身引起的堿基插入或缺失率，從而增強多種crispr堿基編輯器的功效。在進一步描述本發(fā)明具體實施方式之前，應(yīng)理解，本發(fā)明的保護范圍不局限于下述特定的具體實施方案；還應(yīng)當理解，本發(fā)明實施例中使用的術(shù)語是為了描述特定的具體實施方案，而不是為了限制本發(fā)明的保護范圍。除非另外說明，本發(fā)明中所公開的實驗方法、檢測方法、制備方法均采用本
技術(shù)領(lǐng)域：
常規(guī)的分子生物學(xué)、生物化學(xué)、細胞生物學(xué)、重組dna技術(shù)及相關(guān)領(lǐng)域的常規(guī)技術(shù)，這些技術(shù)在現(xiàn)有文獻中已有完善說明。實施例1一種堿基編輯系統(tǒng)，包括ugi表達質(zhì)粒(序列為seqidno：20)和be3表達質(zhì)粒(序列為seqidno：21)，或包括ugi和be3共表達質(zhì)粒。所述的ugi和be3共表達質(zhì)粒為增強型crispr堿基編輯器ebea表達質(zhì)粒(序列為seqidno：22)或增強型crispr堿基編輯器ebeb表達質(zhì)粒(序列為seqidno：23)。人基因組fancf位點利用上述的堿基編輯系統(tǒng)實施高功效高精度的堿基編輯：一.實驗材料1)試劑引物按常規(guī)方法合成自上海博尚生物有限公司；限制性內(nèi)切酶、dna連接酶、高保真dna聚合酶購自neb公司；質(zhì)粒重組試劑盒clone購自vazyme公司；pcmv-be3購自addgene網(wǎng)站，序列為seqidno：21；dna膠回收試劑盒購自coming公司；轉(zhuǎn)染試劑2000，購自thermofisher公司；quickextracttm基因組dna抽提試劑、高通量dna二代測序文庫構(gòu)建試劑盒tmseqchiplibarypreparationkit購自illumina公司。2)細胞株人胎腎細胞hek293ft(thermofisherscientific，r70007)在加入了1ml含有1％雙抗的dmem(gibco，10566)+10％fbs(gbico，16000-044)培養(yǎng)基的6孔板中貼壁培養(yǎng)至單孔細胞個數(shù)為八十萬。二.實驗方法1、堿基編輯系統(tǒng)的構(gòu)建1.1ugi單獨表達質(zhì)粒的構(gòu)建使用以下引物1和含有核定位序列(nls)的引物2將ugi基因從pcmv-be3質(zhì)粒(addgene，#73021)中pcr擴增出來，之后用質(zhì)粒重組試劑盒clone(vazyme，c112-02)按試劑盒操作說明將下游連有nls的ugi基因重組至真核表達載體pcdna3.1(+)(thermofisherscientific，v79020)中，得到ugi單獨表達質(zhì)粒pugi-nls(序列為seqidno：20)。1.2ebea、ebeb表達質(zhì)粒的構(gòu)建利用以下引物3和引物4以pcmv-be3質(zhì)粒(addgene，#73021)為模版進行pcr擴增，將pcr產(chǎn)物用質(zhì)粒重組試劑盒clone(vazyme，c112-02)按試劑盒操作說明重組至pcmv-be3質(zhì)粒(addgene，#73021)中得到含有apai(neb，r0114s)、pmei(neb，r0560l)酶切位點的質(zhì)粒pcmv-be3ap。利用以下引物5和引物6以化學(xué)合成的dna序列(序列為seqidno：19)為模版進行pcr擴增，將pcr產(chǎn)物經(jīng)限制性內(nèi)切酶apai(neb，r0114s)、pmei(neb，r0560l)消化后利用t4dna連接酶(neb，m0202l)按試劑說明連入同樣經(jīng)限制性內(nèi)切酶apai(neb，r0114s)、pmei(neb，r0560l)消化后的pcmv-be3ap(25℃，2hr)，得到ebeb表達質(zhì)粒pcmv-ebeb(序列為seqidno：23)。利用以下引物3，引物7，引物8和引物9以pcmv-be3為模版進行pcr擴增，將兩段pcr產(chǎn)物用質(zhì)粒重組試劑盒clone(vazyme，c112-02)按試劑盒操作說明重組至pcmv-be3質(zhì)粒(addgene，#73021)中得到pcmv-be3t。利用以下引物5和引物10以pcmv-be3t為模版進行pcr擴增，將pcr產(chǎn)物經(jīng)限制性內(nèi)切酶apai(neb，r0114s)、pmei(neb，r0560l)消化后利用t4dna連接酶(neb，m0202l)按試劑說明連入同樣經(jīng)限制性內(nèi)切酶apai(neb，r0114s)、pmei(neb，r0560l)消化后的pcmv-be3ap(25℃，2hr)，得到ebea表達質(zhì)粒pcmv-ebea(序列為seqidno：22)。2、堿基編輯系統(tǒng)的應(yīng)用2.1sgrna表達質(zhì)粒的構(gòu)建將以下引物11和引物12退火，將退火產(chǎn)物利用t4dna連接酶(neb，m0202l)按照試劑說明連入經(jīng)限制性內(nèi)切酶bsai(neb，r3535l)消化后的sgrna表達載體pgl3-u6-sgrna-pgk-puromycin(addgene，#51133)后，得到靶向人基因組fancf位點的sgrna表達質(zhì)粒psgfancf(序列為seqidno：24)。表1引物步驟1.2、1.2和2.1中質(zhì)粒構(gòu)建實驗中擴增各種片段使用pcr體系如下：phusion(neb，m0535l)1μl；5×buffer(neb，b0518s)4μl；forwardprimer(5μm)1μl；reverseprimer(5μm)1μl；template10ng；rnase-freewater補至50μl。pcr程序如下：95℃，2min；95℃，15sec，55℃，30sec，72℃，1min，30循環(huán)；72℃，3min。2.2真核細胞轉(zhuǎn)染2.2.1雙載體共表達be3和ugi將psgfancf、pcmv-be3和得到的序列正確的pugi-nls質(zhì)粒按照1.6μg/2.5μg/0ng(be3_nougi)，1.6μg/2.5μg/50ng(be3_lowugi)，1.6μg/2.5μg/100ng(be3_mediumugi)，1.6μg/2.5μg/200ng(be3_highugi)，0μg/0μg/0ng(nobe3)的比例混合入250μlopti-mem中，再混入含有2000(thermofisher，15338100)轉(zhuǎn)染試劑的250μlopti-mem(thermofisher，51985-042)，室溫放置30分鐘后加入含有1ml1％雙抗的dmem(gibco，10566)+10％fbs(gbico，16000-044)培養(yǎng)基和八十萬hek293ft細胞的6孔板中進行轉(zhuǎn)染。轉(zhuǎn)染24h后換含有1ug/mlpuromycine(invivogen，ant-pr-1)抗生素的新鮮培養(yǎng)基，繼續(xù)培養(yǎng)48小時。2.2.2單載體共表達be3和ugi將psgfancf和pcmv-be3(或得到的序列正確的pcmv-ebea或pcmv-ebeb)質(zhì)粒按照1.6μg/2.5μg的比例混合入250μlopti-mem中，再混入含有2000(thermofisher，15338100)轉(zhuǎn)染試劑的250μlopti-mem(thermofisher，51985-042)，室溫放置30分鐘后加入含有1ml1％雙抗的dmem(gibco，10566)+10％fbs(gbico，16000-044)培養(yǎng)基和八十萬hek293ft細胞的6孔板中進行轉(zhuǎn)染。轉(zhuǎn)染24h后換含有1ug/mlpuromycine(invivogen，ant-pr-1)抗生素的新鮮培養(yǎng)基，繼續(xù)培養(yǎng)48小時。2.3基因組dna抽提和pcr擴增利用quickextracttm(epicenter/illumina，qe09050)試劑按照試劑操作說明抽提轉(zhuǎn)染后hek293ft細胞的基因組dna，將抽提得到的基因組dna利用以下引物13和引物14進行pcr擴增，并利用dna膠回收試劑(ap-gx-500g)按照試劑操作說明對pcr產(chǎn)物進行切膠回收，用于后續(xù)的高通量二代dna測序文庫的構(gòu)建。表2引物編號引物名序列13hfancf_gc1fcacagtatgtctctggcgtt14hfancf_gc1rtctcttggagtgtctcctca擴增基因組dna使用pcr體系如下：phusion(neb，m0535l)1μl；5×buffer(neb，b0518s)4μl；forwardprimer(5μm)1μl；reverseprimer(5μm)1μl；template10ng；rnase-freewater補至50μl。pcr程序如下：95℃，2min；95℃，15sec，55℃，15sec，72℃，30sec，30循環(huán)；72℃，3min。2.4高通量二代dna測序?qū)cr產(chǎn)物進行dna-seq的高通量測序文庫制備。具體來說，首先分別使用qubitfluorometer和aglientbioanalyzer2100對pcr產(chǎn)物進行濃度質(zhì)檢和純度鑒定；質(zhì)檢合格的樣品，再使用covariss220超聲波dna破碎儀將pcr產(chǎn)物進行片段化；然后利用illumina官方試劑盒truseqchipsamplepreparationkit，對不同的pcr產(chǎn)物加上不同生物素標記barcode，進行pcr擴增富集，完成測序文庫的制備，并對已經(jīng)構(gòu)建完成的文庫進行濃度和片段大小質(zhì)檢；最后使用illuminahiseq2500測序儀進行高通量測序。2.5實驗結(jié)果通過將sgfancf表達質(zhì)粒、be3表達質(zhì)粒和ugi表達質(zhì)粒共轉(zhuǎn)染真核生物細胞(圖1a)，使其在靶點基因組dna發(fā)生c-t堿基編輯，并在pcr擴增靶點基因組dna之后，利用高通量二代dna測序檢測sgfancf靶點基因組dna發(fā)生的c-t堿基編輯率和堿基插入或缺失率，最終計算所述系統(tǒng)的堿基編輯功效。和未表達ugi的對照組相比，共表達ugi能夠在sgfancf靶點基因組dna的4個c位點提高堿基編輯器的功效，并且其功效隨著ugi轉(zhuǎn)染量的增加而提高(圖1b)。同時，sgfancf靶點基因組dna的4個c位點的c-t堿基編輯率也得到提高(圖1c)，而crispr系統(tǒng)本身所引起的堿基插入或缺失率則得到抑制(圖1d)。進一步的，本發(fā)明利用t2a自剪切肽段在be3編碼區(qū)下游同框接入1×或者3×ugi基因，得到增強型堿基編輯器a(enhancedbaseeditor，ebea)和增強型堿基編輯器b(ebeb)。在將sgfancf表達質(zhì)粒和be3、ebea或ebeb表達質(zhì)粒共轉(zhuǎn)染真核細胞之后(圖1e)，按照上述方法檢測sgfancf靶點基因組dna發(fā)生的c-t堿基編輯率和堿基插入或缺失率，并計算增強型堿基編輯器的功效。和be3相比，ebea和ebeb的功效均得到提高，并且同框接入3×ugi基因的ebeb功效為最高(圖1f)。同時，ebea和ebeb在sgfancf靶點基因組dna的4個c位點所誘導(dǎo)的c-t堿基編輯率也比be3更高(圖1g)，而crispr系統(tǒng)本身所引起的堿基插入或缺失率則較be3更低(圖1h)。實施例2人基因組rnf2位點利用實施例1中的堿基編輯系統(tǒng)實施高功效高精度的堿基編輯：1.實驗材料1)試劑同實施例1。2)細胞株和載體同實施例1。2.實驗方法1、堿基編輯系統(tǒng)的構(gòu)建1.1ugi單獨表達質(zhì)粒的構(gòu)建同實施例1。1.2ebea、ebeb表達質(zhì)粒的構(gòu)建同實施例1。2、堿基編輯系統(tǒng)的應(yīng)用2.1sgrna表達質(zhì)粒的構(gòu)建將以下引物15和引物16退火，將退火產(chǎn)物利用t4dna連接酶(neb，m0202l)按照試劑說明連入經(jīng)限制性內(nèi)切酶bsai(neb，r3535l)消化后的sgrna表達載體pgl3-u6-sgrna-pgk-puromycin(addgene，#51133)后，得到靶向人基因組rnf2位點的sgrna表達質(zhì)粒psgrnf2(序列為seqidno：25)。表3引物編號引物名序列15fwd_rnf2accggtcatcttagtcattacctg16rev_rnf2aaaccaggtaatgactaagatgac質(zhì)粒構(gòu)建實驗中擴增各種片段使用pcr體系如下：phusion(neb，m0535l)1μl；5×buffer(neb，b0518s)4μl；forwardprimer(5μm)1μl；reverseprimer(5μm)1μl；template10ng；rnase-freewater補至50μl。pcr程序如下：95℃，2min；95℃，15sec，55℃，30sec，72℃，1min，30循環(huán)；72℃，3min。2.2真核細胞轉(zhuǎn)染2.2.1雙載體共表達be3和ugi將psgrnf2、pcmv-be3和得到的序列正確的pugi-nls質(zhì)粒按照1.6μg/2.5μg/0ng(be3_nougi)，1.6μg/2.5μg/50ng(be3_lowugi)，1.6μg/2.5μg/100ng(be3_mediumugi)，1.6μg/2.5μg/200ng(be3_highugi)，0μg/0μg/0ng(nobe3)的比例混合入250μlopti-mem(thermofisher，51985-042)中，再混入含有2000(thermofisher，15338100)轉(zhuǎn)染試劑的250μlopti-mem，室溫放置30分鐘后加入含有1mldmemdmem(gibco，10566)+10％fbs(gbico，16000-044)培養(yǎng)基和八十萬hek293ft細胞的6孔板中進行轉(zhuǎn)染。轉(zhuǎn)染24h后換含有1ug/mlpuromycine(invivogen，ant-pr-1)抗生素的新鮮培養(yǎng)基，繼續(xù)培養(yǎng)48小時。2.2.1雙載體共表達be3和ugi將psgrnf2和pcmv-be3(或得到的序列正確的pcmv-ebea或pcmv-ebeb)質(zhì)粒按照1.6μg/2.5μg的比例混合入250μlopti-mem(thermofisher，51985-042)中，再混入含有2000(thermofisher，15338100)轉(zhuǎn)染試劑的250μlopti-mem，室溫放置30分鐘后加入含有1ml1％雙抗的dmem(gibco，10566)+10％fbs(gbico，16000-044)培養(yǎng)基和八十萬hek293ft細胞的6孔板中進行轉(zhuǎn)染。轉(zhuǎn)染24h后換含有1ug/mlpuromycine(invivogen，ant-pr-1)抗生素的新鮮培養(yǎng)基，繼續(xù)培養(yǎng)48小時。2.3基因組dna抽提和pcr擴增利用quickextracttm試劑按試劑操作說明抽提轉(zhuǎn)染后hek293ft細胞的基因組dna，將抽提得到的基因組dna利用以下引物17和引物18進行pcr擴增，并利用dna膠回收試劑按試劑操作說明對pcr產(chǎn)物進行切膠回收，用于后續(xù)的高通量二代dna測序文庫的構(gòu)建。表4引物編號引物名序列17hrnf2_g2faggaggacttgcccaacttt18hrnf2_g2rctgtgtcagaacatgctggaa擴增基因組dna使用pcr體系如下：phusion(neb，m0535l)1μl；5×buffer(neb，b0518s)4μl；forwardprimer(5μm)1μl；reverseprimer(5μm)1μl；template10ng；rnase-freewater補至50μl。pcr程序如下：95℃，2min；95℃，15sec，55℃，15sec，72℃，30sec，30循環(huán)；72℃，3min。2.4高通量二代dna測序同實施例1。2.5實驗結(jié)果通過將sgrnf2表達質(zhì)粒、be3表達質(zhì)粒和ugi表達質(zhì)粒共轉(zhuǎn)染真核生物細胞(圖2a)，使其在靶點基因組dna發(fā)生c-t堿基編輯，并在pcr擴增靶點基因組dna之后，利用高通量二代dna測序檢測sgrnf2靶點基因組dna發(fā)生的c-t堿基編輯率和堿基插入或缺失率，最終計算所述系統(tǒng)的堿基編輯功效。和未表達ugi的對照組相比，共表達ugi能夠在sgrnf2靶點基因組dna的2個c位點提高堿基編輯器的功效，并且其功效隨著ugi轉(zhuǎn)染量的增加而提高(圖2b)。同時，sgrnf2靶點基因組dna的2個c位點的c-t堿基編輯率也得到提高(圖2c)，而crispr系統(tǒng)本身所引起的堿基插入或缺失率則得到抑制(圖2d)。進一步的，在將sgrnf2表達質(zhì)粒和be3、ebea或ebeb表達質(zhì)粒共轉(zhuǎn)染真核細胞之后(圖2e)，按照上述方法檢測sgrnf2靶點基因組dna發(fā)生的c-t堿基編輯率和堿基插入或缺失率，并計算增強型堿基編輯器的功效。和be3相比，ebea和ebeb的功效均得到提高，并且同框接入3×ugi基因的ebeb功效為最高(圖2f)。同時，ebea和ebeb在sgrnf2靶點基因組dna的2個c位點所誘導(dǎo)的c-t堿基編輯率也比be3更高(圖2g)，而crispr系統(tǒng)本身所引起的堿基插入或缺失率則較be3更低(圖2h)。<110>上?？萍即髮W(xué)<120>一種堿基編輯系統(tǒng)及其構(gòu)建和應(yīng)用方法<130>xxx<160>25<170>patentinversion3.5<210>1<211>42<212>dna<213>artificialsequence<220><223>引物<400>1gtgctggatatctgcagaattcatgactaatctgtcagatat42<210>2<211>59<212>dna<213>artificialsequence<220><223>引物<400>2ttggtaccgagctcggatccctagactttcctcttcttcttggggagcatcttaatctt59<210>3<211>20<212>dna<213>artificialsequence<220><223>引物<400>3aaatggccgaaaacggatgt20<210>4<211>59<212>dna<213>artificialsequence<220><223>引物<400>4gaggctgatcagcgggtttaaactcaatggtgagggcccgactttcctcttcttcttgg59<210>5<211>32<212>dna<213>artificialsequence<220><223>引物<400>5aaagtcgggcccgagggcagaggaagtctgct32<210>6<211>38<212>dna<213>artificialsequence<220><223>引物<400>6cagcgggtttaaactcacacctttcttttttttttagg38<210>7<211>30<212>dna<213>artificialsequence<220><223>引物<400>7agaaccaccagagtcacccccaagctgtga30<210>8<211>89<212>dna<213>artificialsequence<220><223>引物<400>8gactctggtggttctgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaat60cctggcccaactaatctgtcagatattat89<210>9<211>40<212>dna<213>artificialsequence<220><223>引物<400>9cgaggctgatcagcgggtttaaactcaatggtgatggtga40<210>10<211>38<212>dna<213>artificialsequence<220><223>引物<400>10cagcgggtttaaacttagactttcctcttcttcttggg38<210>11<211>23<212>dna<213>artificialsequence<220><223>引物<400>11accggaatcccttctgcagcacc23<210>12<211>23<212>dna<213>artificialsequence<220><223>引物<400>12aaacggtgctgcagaagggattc23<210>13<211>20<212>dna<213>artificialsequence<220><223>引物<400>13cacagtatgtctctggcgtt20<210>14<211>20<212>dna<213>artificialsequence<220><223>引物<400>14tctcttggagtgtctcctca20<210>15<211>24<212>dna<213>artificialsequence<220><223>引物<400>15accggtcatcttagtcattacctg24<210>16<211>24<212>dna<213>artificialsequence<220><223>引物<400>16aaaccaggtaatgactaagatgac24<210>17<211>20<212>dna<213>artificialsequence<220><223>引物<400>17aggaggacttgcccaacttt20<210>18<211>21<212>dna<213>artificialsequence<220><223>引物<400>18ctgtgtcagaacatgctggaa21<210>19<211>1482<212>dna<213>artificialsequence<220><223>化學(xué)合成的pcr模板dna<400>19gctagcgccggagagcttcaaaaggggaacgaactcgcactaccgtctaaatacgtgaat60ttcctgtatttagcgtcccattacgagaagttgaaaggttcacctgaagataacgaacag120aagcaactttttgttgagcagcacaaacattatctcgacgaaatcatagagcaaatttcg180gaattcagtaagagagtcatcctagctgatgccaatctggacaaagtattaagcgcatac240aacaagcacagggataaacccatacgtgagcaggcggaaaatattatccatttgtttact300cttaccaacctcggcgctccagccgcattcaagtattttgacacaacgatagatcgcaaa360cgatacacttctaccaaggaggtgctagacgcgacactgattcaccaatccatcacggga420ttatatgaaactcggatagatttgtcacagcttgggggtgactctggtggttctcccaag480aagaagaggaaagtcgagggcagaggaagtctgctaacatgcggtgacgtcgaggagaat540cctggcccaactaatctgtcagatattattgaaaaggagaccggtaagcaactggttatc600caggaatccatcctcatgctcccagaggaggtggaagaagtcattgggaacaagccggaa660agcgatatactcgtgcacaccgcctacgacgagagcaccgacgagaatgtcatgcttctg720actagcgacgcccctgaatacaagccttgggctctggtcatacaggatagcaacggtgag780aacaagattaagatgctccccaagaagaagaggaaagtcgctactaacttcagcctgctg840aagcaggctggagacgtggaggagaaccctggacctaccaacctgtccgatatcattgag900aaagagaccggcaaacagctggtgatccaggagagcatcctgatgctgcccgaagaggtg960gaggaagtgatcggcaacaagcccgagtccgacatcctggtgcacacagcctatgatgaa1020tccaccgacgagaacgtgatgctgctgacctccgatgctcccgagtataaaccctgggca1080ctggtgatccaggactctaatggagagaacaagatcaagatgctgcccaagaagaagagg1140aaagtcgaaggacggggctccctcctgacctgtggcgatgtggaagagaaccccggcccc1200acaaacctcagtgacattatcgagaaggaaacaggaaaacagctcgtcattcaagaatct1260attcttatgttgcctgaggaagtcgaagaggttattggcaataaacctgaatctgatatt1320cttgtccataccgcttacgatgagtccacagatgaaaatgttatgctgctcacatctgac1380gcaccagagtacaaaccatgggcgctcgttattcaagattccaacggcgaaaacaaaatc1440aaaatgctttctggtggttctcctaaaaaaaaaagaaaggtg1482<210>20<211>5711<212>dna<213>artificialsequence<220><223>ugi表達載體pugi-nls<400>20actcgagcggccgccactgtgctggatatctgcagaattcatgactaatctgtcagatat60tattgaaaaggagaccggtaagcaactggttatccaggaatccatcctcatgctcccaga120ggaggtggaagaagtcattgggaacaagccggaaagcgatatactcgtgcacaccgccta180cgacgagagcaccgacgagaatgtcatgcttctgactagcgacgcccctgaatacaagcc240ttgggctctggtcatacaggatagcaacggtgagaacaagattaagatgctccccaagaa300gaagaggaaagtctagggatccgagctcggtaccaagcttaagtttaaaccgctgatcag360cctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcct420tgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgc480attgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaaggggg540aggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgagg600cggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaa660gcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgc720ccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaag780ctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgacccca840aaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttc900gccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaa960cactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcct1020attggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgt1080gtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcat1140gcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaag1200tatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccat1260cccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttt1320tatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggagg1380cttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcgg1440atctgatcaagagacaggatgaggatcgtttcgcatggaagcgggtaggcctttggggca1500gcggccaatagcagctttgctccttcgctttctgggctcagaggctgggaaggggtgggt1560ccgggggcgggctcaggggcgggctcaggggcggggcgggcgcccgaaggtcctccggag1620gcccggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcat1680ctccgggcctttcgacctgcagcccaagcttaccatgaccgagtacaagcccacggtgcg1740cctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccga1800ctaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagct1860gcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacga1920cggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgc1980cgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagat2040ggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcgg2100cgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtgga2160ggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccc2220cttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcg2280cacctggtgcatgacccgcaagcccggtgcctgagcgggactctggggttcgaaatgacc2340gaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaa2400aggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggat2460ctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaa2520taaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgt2580ggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctag2640agcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaatt2700ccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagc2760taactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgc2820cagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctct2880tccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatca2940gctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaac3000atgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttt3060ttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtgg3120cgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgc3180tctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagc3240gtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctcc3300aagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaac3360tatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggt3420aacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcct3480aactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacc3540ttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttt3600tttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatc3660ttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatg3720agattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatca3780atctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggca3840cctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtag3900ataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagac3960ccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgc4020agaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagct4080agagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatc4140gtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaagg4200cgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatc4260gttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataat4320tctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaag4380tcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggat4440aataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcgggg4500cgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgca4560cccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacagga4620aggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactc4680ttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacata4740tttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtg4800ccacctgacgtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaa4860tctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcg4920ctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgca4980tgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatata5040cgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttc5100atagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgac5160cgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaa5220tagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcag5280tacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggc5340ccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct5400acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtg5460gatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtt5520tgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattga5580cgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaa5640ctagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagaccc5700aagctggctag5711<210>21<211>8532<212>dna<213>artificialsequence<220><223>be3表達載體pcmv-be3<400>21atatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatg60cccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcg120ctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgact180cacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaa240atcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggta300ggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgct360agagatccgcggccgctaatacgactcactatagggagagccgccaccatgagctcagag420actggcccagtggctgtggaccccacattgagacggcggatcgagccccatgagtttgag480gtattcttcgatccgagagagctccgcaaggagacctgcctgctttacgaaattaattgg540gggggccggcactccatttggcgacatacatcacagaacactaacaagcacgtcgaagtc600aacttcatcgagaagttcacgacagaaagatatttctgtccgaacacaaggtgcagcatt660acctggtttctcagctggagcccatgcggcgaatgtagtagggccatcactgaattcctg720tcaaggtatccccacgtcactctgtttatttacatcgcaaggctgtaccaccacgctgac780ccccgcaatcgacaaggcctgcgggatttgatctcttcaggtgtgactatccaaattatg840actgagcaggagtcaggatactgctggagaaactttgtgaattatagcccgagtaatgaa900gcccactggcctaggtatccccatctgtgggtacgactgtacgttcttgaactgtactgc960atcatactgggcctgcctccttgtctcaacattctgagaaggaagcagccacagctgaca1020ttctttaccatcgctcttcagtcttgtcattaccagcgactgcccccacacattctctgg1080gccaccgggttgaaaagcggcagcgagactcccgggacctcagagtccgccacacccgaa1140agtgataaaaagtattctattggtttagccatcggcactaattccgttggatgggctgtc1200ataaccgatgaatacaaagtaccttcaaagaaatttaaggtgttggggaacacagaccgt1260cattcgattaaaaagaatcttatcggtgccctcctattcgatagtggcgaaacggcagag1320gcgactcgcctgaaacgaaccgctcggagaaggtatacacgtcgcaagaaccgaatatgt1380tacttacaagaaatttttagcaatgagatggccaaagttgacgattctttctttcaccgt1440ttggaagagtccttccttgtcgaagaggacaagaaacatgaacggcaccccatctttgga1500aacatagtagatgaggtggcatatcatgaaaagtacccaacgatttatcacctcagaaaa1560aagctagttgactcaactgataaagcggacctgaggttaatctacttggctcttgcccat1620atgataaagttccgtgggcactttctcattgagggtgatctaaatccggacaactcggat1680gtcgacaaactgttcatccagttagtacaaacctataatcagttgtttgaagagaaccct1740ataaatgcaagtggcgtggatgcgaaggctattcttagcgcccgcctctctaaatcccga1800cggctagaaaacctgatcgcacaattacccggagagaagaaaaatgggttgttcggtaac1860cttatagcgctctcactaggcctgacaccaaattttaagtcgaacttcgacttagctgaa1920gatgccaaattgcagcttagtaaggacacgtacgatgacgatctcgacaatctactggca1980caaattggagatcagtatgcggacttatttttggctgccaaaaaccttagcgatgcaatc2040ctcctatctgacatactgagagttaatactgagattaccaaggcgccgttatccgcttca2100atgatcaaaaggtacgatgaacatcaccaagacttgacacttctcaaggccctagtccgt2160cagcaactgcctgagaaatataaggaaatattctttgatcagtcgaaaaacgggtacgca2220ggttatattgacggcggagcgagtcaagaggaattctacaagtttatcaaacccatatta2280gagaagatggatgggacggaagagttgcttgtaaaactcaatcgcgaagatctactgcga2340aagcagcggactttcgacaacggtagcattccacatcaaatccacttaggcgaattgcat2400gctatacttaga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tcatccatagttgcctgactccccgtcgtgtagataacta7140cgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgct7200caccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtg7260gtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaa7320gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgt7380cacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagtta7440catgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtca7500gaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctctta7560ctgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct7620gagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccg7680cgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaac7740tctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaact7800gatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaa7860atgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttccttt7920ttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaat7980gtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctg8040acgtcgacggatcgggagatcgatctcccgatcccctagggtcgactctcagtacaatct8100gctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctg8160agtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatga8220agaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgc8280gttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcata8340gcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgc8400ccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatag8460ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtac8520atcaagtgtatc8532<210>22<211>8848<212>dna<213>artificialsequence<220><223>ebea表達載體pcmv-ebea<400>22atatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatg60cccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcg120ctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgact180cacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaa240atcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggta300ggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgct360agagatccgcggccgctaatacgactcactatagggagagccgccaccatgagctcagag420actggcccagtggctgtggaccccacattgagacggcggatcgagccccatgagtttgag480gtattcttcgatccgagagagctccgcaaggagacctgcctgctttacgaaattaattgg540gggggccggcactccatttggcgacatacatcacagaacactaacaagcacgtcgaagtc600aacttcatcgagaagttcacgacagaaagatatttctgtccgaacacaaggtgcagcatt660acctggtttctcagctggagcccatgcggcgaatgtagtagggccatcactgaattcctg720tcaaggtatccccacgtcactctgtttatttacatcgcaaggctgtaccaccacgctgac780ccccgcaatcgacaaggcctgcgggatttgatctcttcaggtgtgactatccaaattatg840actgagcaggagtcaggatactgctggagaaactttgtgaattatagcccgagtaatgaa900gcccactggcctaggtatccccatctgtgggtacgactgtacgttcttgaactgtactgc960atcatactgggcctgcctccttgtctcaacattctgagaaggaagcagccacagctgaca1020ttctttaccatcgctcttcagtcttgtcattaccagcgactgcccccacacattctctgg1080gccaccgggttgaaaagcggcagcgagactcccgggacctcagagtccgccacacccgaa1140agtgataaaaagtattctattggtttagccatcggcactaattccgttggatgggctgtc1200ataaccgatgaatacaaagtaccttcaaagaaatttaaggtgttggggaacacagaccgt1260cattcgattaaaaagaatcttatcggtgccctcctattcgatagtggcgaaacggcagag1320gcgactcgcctgaaacgaaccgctcggagaaggtatacacgtcgcaagaaccgaatatgt1380tacttacaagaaatttttagcaatgagatggccaaagttgacgattctttctttcaccgt1440ttggaagagtccttccttgtcgaagaggacaagaaacatgaacggcaccccatctttgga1500aacatagtagatgaggtggcatatcatgaaaagtacccaacgatttatcacctcagaaaa1560aagctagttgactcaactgataaagcggacctgaggttaatctacttggctcttgcccat1620atgataaagttccgtgggcactttctcattgagggtgatctaaatccggacaactcggat1680gtcgacaaactgttcatccagttagtacaaacctataatcagttgtttgaagagaaccct1740ataaatgcaagtggcgtggatgcgaaggctattcttagcgcccgcctctctaaatcccga1800cggctagaaaacctgatcgcacaattacccggagagaagaaaaatgggttgttcggtaac1860cttatagcgctctcactaggcctgacaccaaattttaagtcgaacttcgacttagctgaa1920gatgccaaattgcagcttagtaaggacacgtacgatgacgatctcgacaatctactggca1980caaattggagatcagtatgcggacttatttttggctgccaaaaaccttagcgatgcaatc2040ctcctatctgacatactgagagttaatactgagattaccaaggcgccgttatccgcttca2100atgatcaaaaggtacgatgaacatcaccaagacttgacacttctcaaggccctagtccgt2160cagcaactgcctgagaaatataaggaaatattctttgatcagtcgaaaaacgggtacgca2220ggttatattgacggcggagcgagtcaagaggaattctacaagtttatcaaacccatatta2280gagaagatggatgggacggaagagttgcttgtaaaactcaatcgcgaagatctactgcga2340aagcagcggactttcgacaacggtagcattccacatcaaatccacttaggcgaattgcat2400gctatacttagaaggcaggaggatttttatccgttcctcaaagacaatcgtgaaaagatt2460gagaaaatcctaacctttcgcataccttactatgtgggacccctggcccgagggaactct2520cggttcgcatggatgacaagaaagtccgaagaaacgattactccatggaattttgaggaa2580gttgtcgataaaggtgcgtcagctcaatcgttcatcgagaggatgaccaactttgacaag2640aatttaccgaacgaaaaagtattgcctaagcacagtttactttacgagtatttcacagtg2700tacaatgaactcacgaaagttaagtatgtcactgagggcatgcgtaaacccgcctttcta2760agcggagaacagaagaaagcaatagtagatctgttattcaagaccaaccgcaaagtgaca2820gttaagcaattgaaagaggactactttaagaaaattgaatgcttcgattctgtcgagatc2880tccggggtagaagatcgatttaatgcgtcacttggtacgtatcatgacctcctaaagata2940attaaagataaggacttcctggataacgaagagaatgaagatatcttagaagatatagtg3000ttgactcttaccctctttgaagatcgggaaatgattgaggaaagactaaaaacatacgct3060cacctgttcgacgataaggttatgaaacagttaaagaggcgtcgctatacgggctgggga3120cgattgtcgcggaaacttatcaacgggataagagacaagcaaagtggtaaaactattctc3180gattttctaaag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t3480aagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggt3540gtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagt3600tacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgt3660cagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctct3720tactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcatt3780ctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataatac3840cgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaa3900actctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaa3960ctgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggca4020aaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcct4080ttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttga4140atgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacc4200tgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgac4260cgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgc4320cacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatt4380tagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgg4440gccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatag4500tggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgattt4560ataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatt4620taacgcgaattttaacaaaatattaacgtttacaatttcccattcgccattcaggctgcg4680caactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctacc4740atgataagtaagtaatattaaggtacgggaggtacttggagcggccgcaataaaatatct4800ttattttcattacatctgtgtgttggttttttgtgtgaatcgatagtactaacatacgct4860ctccatcaaaacaaaacgaaacaaaacaaactagcaaaataggctgtccccagtgcaagt4920gcaggtgccagaacatttctctatcgata4949<210>25<211>4950<212>dna<213>artificialsequence<220><223>sgrnf2表達載體<400>25ggtaccgattagtgaacggatctcgacggtatcgatcacgagactagcctcgagcggccg60cccccttcaccgagggcctatttcccatgattccttcatatttgcatatacgatacaagg120ctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaata180cgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaa240tggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatct300tgtggaaaggacgaaacaccggtcatcttagtcattacctggttttagagctagaaatag360caagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttt420ttttaaagaattctcgacctcgagacaaatggcagtattcatccacaattttaaaagaaa480aggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacat540acaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacag600ggacagcagagatccactttggccgcggctcgagggggttggggttgcgccttttccaag660gcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcg720gcgccgaccctgggactcgcacattcttcacgtccgttcgcagcgtcacccggatcttcg780ccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggtt840ccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccc900tcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggcca960atagcggctgctcagcagggcgcgccgagagcagcggccgggaaggggcggtgcgggagg1020cggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgca1080agcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctc1140cccagggggatccaccggagcttaccatgaccgagtacaagcccacggtgcgcctcgcca1200cccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccg1260ccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaac1320tcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccg1380cggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcg1440gcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcc1500tcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgc1560ccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccg1620agcgcgccggggtgcccgccttcctggaaacctccgcgccccgcaacctccccttctacg1680agcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggt1740gcatgacccgcaagcccggtgcctgacgcccgccccacgacccgcagcgcccgaccgaaa1800ggagcgcacgaccccatgcatcggtacctttaagaccaatgacttacaaggcagctgtag1860atcttagccactttctagagtcggggcggccggccgcttcgagcagacatgataagatac1920attgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaa1980atttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaac2040aacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagc2100aagtaaaacctctacaaatgtggtaaaatcgataaggatccgtcgaccgatgcccttgag2160agccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcact2220tatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctcttccgctt2280cctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcact2340caaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgag2400caaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccata2460ggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacc2520cgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctg2580ttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgc2640tttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgg2700gctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtc2760ttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacagga2820ttagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacg2880gctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaa2940aaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttg3000tttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctttt3060ctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagat3120tatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatct3180aaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcaccta3240tctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataa3300ctacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccac3360gctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaa3420gtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagag3480taagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtgg3540tgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgag3600ttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttg3660tcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctc3720ttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcat3780tctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataata3840ccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaa3900aactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcaccca3960actgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggc4020aaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcc4080tttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttg4140aatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac4200ctgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtga4260ccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcg4320ccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgat4380ttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtg4440ggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaata4500gtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatt4560tataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaat4620ttaacgcgaattttaacaaaatattaacgtttacaatttcccattcgccattcaggctgc4680gcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagcccaagctac4740catgataagtaagtaatattaaggtacgggaggtacttggagcggccgcaataaaatatc4800tttattttcattacatctgtgtgttggttttttgtgtgaatcgatagtactaacatacgc4860tctccatcaaaacaaaacgaaacaaaacaaactagcaaaataggctgtccccagtgcaag4920tgcaggtgccagaacatttctctatcgata4950當前第1頁12