CN111518220A - 一种融合蛋白及其设计方法 - Google Patents
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Abstract
本发明公开一种融合蛋白及其设计方法。本发明所述设计方法是将具有DNA结合功能的重组辅助蛋白与具有DNA切割功能的蛋白融合；在具有DNA切割功能的融合蛋白切割的同时，重组辅助蛋白在切割位点附近提供DNA重组修复模板，招募重组修复酶，从而大幅地提高了重组修复效率。本发明的优点是重组修复效率提高。
Description
技术领域
本发明涉及生物技术领域，具体地说是一种融合蛋白及其设计方法。
背景技术
Cas9基因编辑技术已经广泛应用于各种生物以及离体培养的细胞的基因编辑。目前常规的方法为Cas9蛋白对基因组DNA定点切割后，通常由生物或者细胞自身的修复系统完成对DNA断裂点的修复。修复方法有不精确的邻端连接修复和精确的重组修复。在动物细胞中，基因组DNA断裂后的修复方式以邻端连接修复为主，重组修复的比例很少，造成重组基因编辑的效率低，需要筛选大量克隆才能得到正确编辑的细胞。
因此需要一种Cas9融合蛋白以及设计类似蛋白的方法。设计出的类似蛋白采用将具有DNA结合功能的重组辅助蛋白与具有DNA切割功能的Cas9蛋白融合。在Cas9切割的同时，重组辅助蛋白在切割位点附近提供DNA重组修复模板，招募重组修复酶，从而极大地提高了重组修复效率。
融合蛋白技术是为获得大量标准融合蛋白而进行的有目的性的基因融合和蛋白表达方法，利用融合蛋白技术，可构建和表达具有多种功能的新型目的蛋白。
发明内容
本发明的一个目的在于提出一类融合蛋白，另一个目的在于提出一种融合蛋白的设计方法。本发明采用将具有特异性DNA切割功能的Cas9蛋白与具有DNA 结合功能的重组辅助蛋白融合。在Cas9切割的同时，重组辅助蛋白在切割位点附近提供DNA重组修复模板，招募重组修复酶，从而极大地提高了重组修复效率。
为实现上述目的，本发明所述一种融合蛋白由两个蛋白功能域连接组成；所述两个蛋白功能域分别为具有特异性DNA切割功能的蛋白功能域和具有DNA 结合功能的重组辅助蛋白功能域。
所述两个蛋白功能域通过多肽、化学键、配体亲和连接或者其他物理、化学方法连接组成。
所述在具有特异性DNA切割功能的蛋白功能域的氮端或者碳端连接具有DNA 结合功能的重组辅助蛋白功能域。
所述具有特异性DNA切割功能的蛋白功能域为具有可编程、特异性DNA切割功能的蛋白功能域，所述具有特异性DNA切割功能的蛋白功能域为Cas9蛋白、 Cpf1蛋白，及其变体中的一种。
所述具有DNA结合功能的重组辅助蛋白功能域为具有DNA结合功能的重组修复辅助蛋白功能域或者具有DNA结合功能的蛋白功能域，具有DNA结合功能的重组辅助蛋白功能域为Rad51蛋白、Rad51同源蛋白、Rad52蛋白、Rad52同源蛋白、RecA蛋白、RecA同源蛋白中的一种。
所述同源蛋白为氨基酸序列具有明显的相似性，在不同生物体或同一机体内行使相同或相似功能的蛋白质。
所述的一种融合蛋白设计方法，在有可编程、特异性DNA切割功能的蛋白域的氮端或者碳端添加具有DNA结合功能的重组修复辅助蛋白或者其DNA结合功能的重组蛋白域。
所述本发明方法设计出的融合蛋白通过将该融合蛋白与重组模板DNA、特异性引导RNA一起转入细胞或者生物体发挥作用。
所述本发明方法设计出的融合蛋白该融合蛋白的应用包括细胞或者生物体的基因组编辑，包括且不限于基因敲除、基因敲入以基因定点突变。
所述的一种融合蛋白设计方法，实现步骤如下：
1、融合蛋白由具有特异性DNA切割功能的蛋白功能域和具有DNA结合功能的重组辅助蛋白功能域组成；
2、两个蛋白功能域之间由多肽、化学键、配体受体亲和连接；
3、具有特异性DNA切割功能的蛋白功能域和具有DNA结合功能的重组辅助蛋白功能域的顺序为具有特异性DNA切割功能的蛋白功能域-具有DNA结合功能的重组辅助蛋白功能域。
所述的融合蛋白的编码序列采用基因合成或者其他常规分子生物学方法获得。
所述的融合蛋白采用大肠杆菌表达系统、枯草芽孢杆菌表达系统、酵母表达系统、动物细胞表达系统以及无细胞表达系统等多种蛋白表达合成方法获得。
本发明所述一种融合蛋白及其设计方法，其有益效果在于：本发明所述用本设计方法设计出的融合蛋白由具有可编程、特异性DNA切割功能的蛋白域和具有DNA结合功能的重组修复辅助蛋白或者其DNA结合功能结构域组成；在具有可编程、特异性DNA切割功能的蛋白域切割的同时，具有DNA结合功能的重组修复辅助蛋白或者其DNA结合功能结构域在切割位点附近提供DNA重组修复模板，招募重组修复酶，从而极大地提高了重组修复效率。
附图说明
图1为本发明促进重组修复原理示意图；
图2为本发明在稳定整合mcherry荧光蛋白的293T细胞中敲入GFP基因示意图。
具体实施方式
实施例1
融合蛋白1：spCas9-Rad51(H)的设计方法以及用大肠杆菌表达宿主表达和纯化spCas9-Rad51(H)蛋白。
融合蛋白1：spCas9-Rad51(H)的设计方法。
1、具有特异性DNA切割功能的蛋白功能域选择spCas9蛋白；
2、具有DNA结合功能的重组辅助蛋白功能域选择人Rad51蛋白；
3、二者之间用多肽GGGGSGGSGGSGGGS连接；
4、蛋白结构为spCas9-连接肽-Rad51(H)。
采用大肠杆菌原核表达系统融合表达spCas9-Rad51(H)的方法，实现步骤如下：
1、将人工合成的spCas9-Rad51(H)编码序列，连接到原核表达载体pET-30a，获得表达载体pET-spCas9-Rad51(H)；
2、转化大肠杆菌表达菌株BL21(DE3)，转化方法如下：
1)取BL21电转感受态，冰上融化；
2)加入表达载体pET-spCas9-Rad51(H)20ng；
3)电击转化，电击条件1.5kV 5ms；
4)加入1ml培养基，培养1h；
5)筛选阳性克隆株；
3、spCas9-R Rad51(H)表达菌株表达蛋白：
1)将spCas9-Rad51(H)表达菌株接种培养基进行培养；
2)当菌株生长到生长对数中期，加入IPTG至终浓度为0.1mM，诱导蛋白表达；
3)4℃ 4000g离心收集菌体；
4、spCas9-Rad51(H)蛋白纯化：
1)磷酸盐缓冲溶液重悬菌体，超声裂解菌体；
2)4℃ 16000g离心30min收集上清；
3)镍-琼脂糖凝胶特异性亲和吸附spCas9-Rad51(H)蛋白；咪唑线性梯度洗脱目标蛋白。
实施例2
融合蛋白2：spCas9-Rad52(H)的设计方法，具体实现步骤如下：
1、具有特异性DNA切割功能的蛋白功能域选择spCas9蛋白；
2、具有DNA结合功能的重组辅助蛋白功能域选择人Rad52蛋白；
3、二者之间用多肽GGGGSGGSGGSGGGS连接；
4、蛋白结构为spCas9-连接肽-Rad52(H)。
spCas9-Rad52(H)蛋白的表达和纯化方法同实施例1。
实施例3
融合蛋白3：Rad51(H)-spCas9的设计方法，具体实现步骤如下：
1、具有特异性DNA切割功能的蛋白功能域选择spCas9蛋白；
2、具有DNA结合功能的重组辅助蛋白功能域选择人Rad51蛋白；
3、二者之间用多肽GGGGSGGSGGSGGGS连接；
4、蛋白结构为Rad51(H)-连接肽-spCas9。
Rad51(H)-spCas9蛋白的表达和纯化方法同实施例1。
实施例4
融合蛋白4：Rad52(H)-spCas9的设计方法，具体实现步骤如下：
1、具有特异性DNA切割功能的蛋白功能域选择spCas9蛋白；
2、具有DNA结合功能的重组辅助蛋白功能域选择人Rad52蛋白；
3、二者之间用多肽GGGGSGGSGGSGGGS连接；
4、蛋白结构为Rad52(H)-连接肽-spCas9。
Rad52(H)-spCas9蛋白的表达和纯化方法同实施例1。
实施例5
融合蛋白5：spCas9-Rad51(H)的设计方法，实现步骤如下：
1、具有特异性DNA切割功能的蛋白功能域选择spCas9蛋白；
2、具有DNA结合功能的重组辅助蛋白功能域为酿酒酵母Rad51蛋白；
3、二者之间用多肽GGGGSGGSGGSGGGS连接；
4、蛋白结构为spCas9-连接肽-Rad51(S)。
采用酵母表达系统表达spCas9-Rad51(S)的方法，实现步骤如下：
1、将人工合成的spCas9-Rad51(S)编码序列，连接到表达载体pGAPZ，获得表达载体pGAPZ-spCas9-Rad51(S)；
2、转化毕赤酵母表达菌种GS115，转化方法如下：
1)BglII线性化pGAPZ-spCas9-Rad51(S)载体；
2)取GS115电转感受态，冰上融化；
2)将线性化pGAPZ-spCas9-Rad51(S)100ng加入感受态细胞；
3)电击转化，电击条件1.5kV 10ms；
4)加入1ml培养基，培养1h；
5)涂Zeocyn抗性平板筛选阳性克隆株；
3、spCas9-Rad51(S)表达菌株表达蛋白：
1)将spCas9-Rad51(S)表达菌株接种10ml YPD培养基进行30℃过夜培养；
2)将1ml培养产物接种到250ml YPD培养基进行培养72h；
3)4℃ 4000g离心收集菌体；
4、spCas9-Rad51(S)蛋白纯化：
1)磷酸盐缓冲溶液重悬菌体，超声裂解菌体；
2)4℃ 16000g离心30min收集上清；
3)镍-琼脂糖凝胶特异性亲和吸附spCas9-Rad51(S)蛋白；咪唑线性梯度洗脱目标蛋白。
实施例6
融合蛋白6：spCas9-Rad52(S)的设计方法，具体实现步骤如下：
1、具有特异性DNA切割功能的蛋白功能域选择spCas9蛋白；
2、具有DNA结合功能的重组辅助蛋白功能域选择酿酒酵母Rad52蛋白；
3、二者之间用多肽GGGGSGGSGGSGGGS连接；
4、蛋白结构为spCas9-连接肽-Rad52(S)。
spCas9-Rad52(S)蛋白的表达和纯化方法同实施例5。
实施例7
融合蛋白7：Rad51(S)-spCas9的设计方法，具体实现步骤如下：
1、具有特异性DNA切割功能的蛋白功能域选择spCas9蛋白；
2、具有DNA结合功能的重组辅助蛋白功能域选择酿酒酵母Rad51蛋白；
3、二者之间用多肽GGGGSGGSGGSGGGS连接；
4、蛋白结构为Rad51(S)-连接肽-spCas9。
Rad51(S)-spCas9蛋白的表达和纯化方法同实施例5。
实施例8
融合蛋白8：Rad52(S)-spCas9的设计方法，具体实现步骤如下：
1、具有特异性DNA切割功能的蛋白功能域选择spCas9蛋白；
2、具有DNA结合功能的重组辅助蛋白功能域选择酿酒酵母Rad52蛋白；
3、二者之间用多肽GGGGSGGSGGSGGGS连接；
4、蛋白结构为Rad52(S)-连接肽-spCas9。
Rad52(S)-spCas9蛋白的表达和纯化方法同实施例5。
实施例9
采用实施例1-8中的8种融合蛋白和野生型spCas9蛋白对细胞基因组进行基因敲入。采用类似的方法和实验步骤也可以实现生物体或者细胞的基因敲除、基因敲入或者定点突变。
实验原理为：
1、293-mito-mcherry细胞株是稳定表达线粒体定位mcherry荧光蛋白的 HEK293T细胞株；在mcherry基因编码序列的3’端设计了基因编辑靶点(靶点序列见DNA序列1)；
2、Cas9特异切割位于mcherry C端的靶点，重组片段上有GFP基因；
3、如果重组成功，细胞将表达mcherry和GFP融合蛋白，体现为红色和绿色荧光共同定位于细胞线粒体；
4、如果非特异重组，细胞表达的GFP蛋白将弥散分布，和红色荧光蛋白分布不一致。实验原理如图2所示。
实验步骤如下：
1、合成靶点sgRNA；化学合成靶点sgRNA序列(RNA序列2)；
2、合成ssDNA重组模板(重组模板序列)；
3、将重组模板5ug、sgRNA0.5ug、Cas9融合蛋白或者野生型Cas9蛋白5ug 混合，室温孵育10min；
4、生长旺盛的293-mito-mcherry细胞消化，用电转缓冲液重悬细胞；细胞浓度4×10e7/ml；
5、将Cas9融合蛋白混合物加入200ul细胞悬液中，混匀；
6、200V 50ms电击转化；
7、将转化产物接种到细胞培养皿中培养；
8、抗稻瘟菌素筛选3周；计数细胞克隆数量及绿色荧光和红色荧光共定位细胞数量，计算细胞重组阳性百分比。
实验结果：如表1
实验结果显示，采用Cas9融合蛋白获得的克隆数量是野生型spCas9蛋白的10倍到30倍。阳性率(正确整合的概率)提高了20％。该蛋白极大提高了细胞基因编辑成功率。
表1：
氨基酸序列spCas9：
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD
氨基酸序列Rad51(H)：
mamqmqleanadtsveeesfgpqpisrleqcginandvkkleeagfhtveavayapkkel inikgiseakadkiltesrsvarlecnsvilvyctlrlsgssdspasasrvvgttggiet gsitemfgefrtgktqichtlavtcqlpidrgggegkamyidtegtfrperllavaeryg lsgsdvldnvayarafntdhqtqllyqasammvesryallivdsatalyrtdysgrgels arqmhlarflrmllrladefgvavvitnqvvaqvdgaamfaadpkkpiggniiahasttr lylrkgrgetrickiydspclpeaeamfainadgvgdakd
氨基酸序列Rad51(S)：
msqvqeqhisesqlqygngslmstvpadlsqsvvdgngngssedieatngsgdggglqeq aeaqgemedeaydeaalgsfvpieklqvngitmadvkklresglhtaeavayaprkdlle ikgiseakadkllneaarlvpmgfvtaadfhmrrseliclttgsknldtllgggvetgsi telfgefrtgksqlchtlavtcqipldigggegkclyidtegtfrpvrlvsiaqrfgldp ddalnnvayaraynadhqlrlldaaaqmmsesrfslivvdsvmalyrtdfsgrgelsarq mhlakfmralqrladqfgvavvvtnqvvaqvdggmafnpdpkkpiggnimahssttrlgf kkgkgcqrlckvvdspclpeaecvfaiyedgvgdpreede
氨基酸序列Rad52(H)：
msgteeailggrdshpaagggsvlcfgqcqytaeeyqaiqkalrqrlgpeyissrmaggg qkvcyieghrvinlanemfgyngwahsitqqnvdfvdlnngkfyvgvcafvrvqlkdgsy hedvgygvseglkskalslekarkeavtdglkralrsfgnalgncildkdylrslnklpr qlplevdltkakrqdlepsveearynscrpnmalghpqlqqvtspsrpshavipadqdcs srslsssaveseathqrklrqkqlqqqfrermekqqvrvstpsaekseaappappvthst pvtvsepllekdflagvtqeliktlednsekwavtpdagdgvvkpssradpaqtsdtlal nnqmvtqnrtphsvchqkpqaksgswdlqtysadqrttgnweshrksqdmkkrkydps
氨基酸序列Rad52(S)：
mneimdmdekkpvfgnhsediqtkldkklgpeyiskrvgfgtsriayiegwrvinlanqi fgyngwstevksvvidflderqgkfsigctaivrvtltsgtyredigygtvenerrkpaa ferakksavtdalkrslrgfgnalgnclydkdflakidkvkfdppdfdennlfrptdeis essrtntlhenqeqqqypnkrrqltkvtntnpdstknlvkientvsrgtpmmaapaeans knssnkdtdlksldaskqdqddllddslmfsddfqdddlinmgntnsnvlttekdpvvak qsptassnpeaeqitfvtakaatsvqneryigeesifdpkyqaqsirhtvdqttskhipa svlkdktmttardsvyekfapkgkqlsmknndkelgphmlegagnqvprettpiktnata fppaaaprfappskvvhpngngavpavpqqrstrrevgrpkinplharkpt
连接肽序列：
GGGGSGGSGGSGGGS
氨基酸序列Cas9-Rad51(H)：
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD
GGGGSGGSGGSGGGS
mamqmqleanadtsveeesfgpqpisrleqcginandvkkleeagfhtveavayapkkel inikgiseakadkiltesrsvarlecnsvilvyctlrlsgssdspasasrvvgttggiet gsitemfgefrtgktqichtlavtcqlpidrgggegkamyidtegtfrperllavaeryg lsgsdvldnvayarafntdhqtqllyqasammvesryallivdsatalyrtdysgrgels arqmhlarflrmllrladefgvavvitnqvvaqvdgaamfaadpkkpiggniiahasttr lylrkgrgetrickiydspclpeaeamfainadgvgdakd
氨基酸序列Cas9-Rad51(S)：
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD
GGGGSGGSGGSGGGS
msqvqeqhisesqlqygngslmstvpadlsqsvvdgngngssedieatngsgdggglqeq aeaqgemedeaydeaalgsfvpieklqvngitmadvkklresglhtaeavayaprkdlle ikgiseakadkllneaarlvpmgfvtaadfhmrrseliclttgsknldtllgggvetgsi telfgefrtgksqlchtlavtcqipldigggegkclyidtegtfrpvrlvsiaqrfgldp ddalnnvayaraynadhqlrlldaaaqmmsesrfslivvdsvmalyrtdfsgrgelsarq mhlakfmralqrladqfgvavvvtnqvvaqvdggmafnpdpkkpiggnimahssttrlgf kkgkgcqrlckvvdspclpeaecvfaiyedgvgdpreede
氨基酸序列Cas9-Rad52(H)：
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD
GGGGSGGSGGSGGGS
msgteeailggrdshpaagggsvlcfgqcqytaeeyqaiqkalrqrlgpeyissrmaggg qkvcyieghrvinlanemfgyngwahsitqqnvdfvdlnngkfyvgvcafvrvqlkdgsy hedvgygvseglkskalslekarkeavtdglkralrsfgnalgncildkdylrslnklpr qlplevdltkakrqdlepsveearynscrpnmalghpqlqqvtspsrpshavipadqdcs srslsssaveseathqrklrqkqlqqqfrermekqqvrvstpsaekseaappappvthst pvtvsepllekdflagvtqeliktlednsekwavtpdagdgvvkpssradpaqtsdtlal nnqmvtqnrtphsvchqkpqaksgswdlqtysadqrttgnweshrksqdmkkrkydps
氨基酸序列Cas9-Rad52(S)：
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD
GGGGSGGSGGSGGGS
mneimdmdekkpvfgnhsediqtkldkklgpeyiskrvgfgtsriayiegwrvinlanqi fgyngwstevksvvidflderqgkfsigctaivrvtltsgtyredigygtvenerrkpaa ferakksavtdalkrslrgfgnalgnclydkdflakidkvkfdppdfdennlfrptdeis essrtntlhenqeqqqypnkrrqltkvtntnpdstknlvkientvsrgtpmmaapaeans knssnkdtdlksldaskqdqddllddslmfsddfqdddlinmgntnsnvlttekdpvvak qsptassnpeaeqitfvtakaatsvqneryigeesifdpkyqaqsirhtvdqttskhipa svlkdktmttardsvyekfapkgkqlsmknndkelgphmlegagnqvprettpiktnata fppaaaprfappskvvhpngngavpavpqqrstrrevgrpkinplharkpt
氨基酸序列Rad51(H)-Cas9：
mamqmqleanadtsveeesfgpqpisrleqcginandvkkleeagfhtveavayapkkel inikgiseakadkiltesrsvarlecnsvilvyctlrlsgssdspasasrvvgttggiet gsitemfgefrtgktqichtlavtcqlpidrgggegkamyidtegtfrperllavaeryg lsgsdvldnvayarafntdhqtqllyqasammvesryallivdsatalyrtdysgrgels arqmhlarflrmllrladefgvavvitnqvvaqvdgaamfaadpkkpiggniiahasttr Lylrkgrgetrickiydspclpeaeamfainadgvgdakd
GGGGSGGSGGSGGGS
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD
氨基酸序列Rad51(S)-Cas9：
msqvqeqhisesqlqygngslmstvpadlsqsvvdgngngssedieatngsgdggglqeq aeaqgemedeaydeaalgsfvpieklqvngitmadvkklresglhtaeavayaprkdlle ikgiseakadkllneaarlvpmgfvtaadfhmrrseliclttgsknldtllgggvetgsi telfgefrtgksqlchtlavtcqipldigggegkclyidtegtfrpvrlvsiaqrfgldp ddalnnvayaraynadhqlrlldaaaqmmsesrfslivvdsvmalyrtdfsgrgelsarq mhlakfmralqrladqfgvavvvtnqvvaqvdggmafnpdpkkpiggnimahssttrlgf kkgkgcqrlckvvdspclpeaecvfaiyedgvgdpreede
GGGGSGGSGGSGGGS
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD
氨基酸序列Rad52(H)-Cas9：
msgteeailggrdshpaagggsvlcfgqcqytaeeyqaiqkalrqrlgpeyissrmaggg qkvcyieghrvinlanemfgyngwahsitqqnvdfvdlnngkfyvgvcafvrvqlkdgsy hedvgygvseglkskalslekarkeavtdglkralrsfgnalgncildkdylrslnklpr qlplevdltkakrqdlepsveearynscrpnmalghpqlqqvtspsrpshavipadqdcs srslsssaveseathqrklrqkqlqqqfrermekqqvrvstpsaekseaappappvthst pvtvsepllekdflagvtqeliktlednsekwavtpdagdgvvkpssradpaqtsdtlal nnqmvtqnrtphsvchqkpqaksgswdlqtysadqrttgnweshrksqdmkkrkydps
GGGGSGGSGGSGGGS
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD
氨基酸序列Rad52(S)-Cas9：
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLSQLGGD
GGGGSGGSGGSGGGS
mneimdmdekkpvfgnhsediqtkldkklgpeyiskrvgfgtsriayiegwrvinlanqi fgyngwstevksvvidflderqgkfsigctaivrvtltsgtyredigygtvenerrkpaa ferakksavtdalkrslrgfgnalgnclydkdflakidkvkfdppdfdennlfrptdeis essrtntlhenqeqqqypnkrrqltkvtntnpdstknlvkientvsrgtpmmaapaeans knssnkdtdlksldaskqdqddllddslmfsddfqdddlinmgntnsnvlttekdpvvak qsptassnpeaeqitfvtakaatsvqneryigeesifdpkyqaqsirhtvdqttskhipa svlkdktmttardsvyekfapkgkqlsmknndkelgphmlegagnqvprettpiktnata fppaaaprfappskvvhpngngavpavpqqrstrrevgrpkinplharkpt
DNA序列1靶点序列：
Catggacgagctgtacaagtagg
RNA序列2sgRNA序列：
caUggacgagcUgUacaagUgUUUCagagcUaUGCUGGAAACAGCAUagcaagUUGaaaUaaggcUagUccgUUaUcaacUUgaaaaagUggcaccgagUcggUgcUU
DNA序列2重组模板序列：
tacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcct ccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggcccc gtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccct gaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagacca cctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacc tcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcgg catggacgagctgtacaagTCCAGCGGAGGTcgcaaaggggaggagctgtttactggagtggttccaa tcctggtggagctcgatggcgacgtcaacggccacaaatttagcgtgcgtggtgagggcgagggcgat gccactaatggcaagttaaccctcaagtttatctgtacaacaggcaagctgcccgttccctggcctac cCTCgtcaccacactcacttatggagtgcaatgcttcgctcgttatcccgaccacatgaagcagcacg attttttcaagtccgcgatgcccgagggctacgttcaagaacgcactataagttttaaggacgacggg acatataagacccgtgccgaggtgaagtttgaaggcgatactttggtgaacagaattgagctgaaggg gatcgactttaaagaagacggaaatatcctgggacacaagctggagtataacttcaatagccataacg tttacattacagctgacaaacagaaaaatggtataaaagccaacttcaagatccggcataatgttgaa gatggcagcgtgcagctggctgatcactaccagcagaatactccaatcggcgacgggcccgttctctt gccggataatcactacctgagcacacaatctgtgttgagcaaggaccccaatgaaaagagagaccaca tggtcctcctggagttcgtcacagcagccgggattacacatggtatggatgaactctacaaaggctcc ggagagggccgtggctctttgctgacatgcggagacgttgaagaaaaccctggccccATGCAGGCCAA GCCACTGTCTCAGGAGGAAAGCACACTGATCGAGCGCGCTACTGCCACCATTAACTCCATCCCCATTT CCGAGGACTACAGCGTTGCCTCCGCCGCCCTGTCATCAGACGGGCGCATTTTCACAGGGGTGAACGTG TATCACTTTACCGGGGGGCCCTGTGCTGAACTCGTTGTGCTCGGAACTGCTGCTGCTGCCGCCGCCGG CAACCTGACCTGCATCGTCGCAATTGGAAATGAAAACAGGGGCATTTTATCTCCTTGCGGACGCTGTC GGCAGGTTCTGCTCGACTTGCACCCCGGTATCAAGGCAATTGTTAAGGACAGCGACGGGCAGCCAACC GCCGTGGGCATCCGGGAGCTGCTCCCCTCTGGATACGTCTGGGAGGGCTGAagtaggggccgcgactc tagataattctaccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccc cgctgggcacttggcgctacacaagtggcctctggcctcgcacacattccacatccaccggtaggcgc caaccggctccgttctttggtggccccttcgcgccaccttctactcctcccctagtcaggaagttccc ccccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgc agatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcggc。
Claims (7)
1.一种融合蛋白，其特征在于：所述融合蛋白由两个蛋白功能域连接组成；所述两个蛋白功能域分别为具有特异性DNA切割功能的蛋白功能域和具有DNA结合功能的重组辅助蛋白功能域。
2.如权利要求1所述的一种融合蛋白，其特征在于：所述在具有特异性DNA切割功能的蛋白功能域的氮端或者碳端连接具有DNA结合功能的重组辅助蛋白功能域。
3.如权利要求2所述的一种融合蛋白，其特征在于：所述两个蛋白功能域通过多肽、化学键、配体亲和连接或者其他物理、化学方法连接组成。
4.如权利要求3所述的一种融合蛋白，其特征在于：所述具有特异性DNA切割功能的蛋白功能域为具有可编程、特异性DNA切割功能的蛋白功能域，所述具有特异性DNA切割功能的蛋白功能域为Cas9蛋白、Cpf1蛋白，及其变体中的一种。
5.如权利要求1所述的一种融合蛋白，其特征在于：所述具有DNA结合功能的重组辅助蛋白功能域为具有DNA结合功能的重组修复辅助蛋白功能域或者具有DNA结合功能的蛋白功能域，具有DNA结合功能的重组辅助蛋白功能域为Rad51蛋白、Rad51同源蛋白、Rad52蛋白、Rad52同源蛋白、RecA蛋白、RecA同源蛋白中的一种。
6.权利要求1-5任一所述的一种融合蛋白的应用，其特征在于：所述本发明方法设计出的融合蛋白通过将该融合蛋白与重组模板DNA、特异性引导RNA一起转入细胞或者生物体发挥作用。
7.权利要求6所述的一种融合蛋白的应用，其特征在于：所述应用该融合蛋白对生物基因组DNA对细胞或者生物体基因组DNA进行改变，包括且不限于敲除、敲入或者突变。
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Application publication date: 20200811 |