【技术实现步骤摘要】
【国外来华专利技术】用于增加CRISPR/Cas12f1系统的效率的经工程化的引导RNA及其用途
[0001][相关申请][0002]本申请要求于2020年10月8日提交的韩国专利申请No.10
‑
2020
‑
0129937和2021年4月21日提交的韩国专利申请No.10
‑
2021
‑
0051552的优先权,以引用方式将其各自的公开内容整体并入本文。
[0003][0004][
][0005]本文公开了使用CRISPR/Cas系统、特别是CRISPR/Cas12f1系统用于基因编辑的领域的技术。
[0006]
技术介绍
[0007]CRISPR/Cas12f1系统是一种被归类为2类V型的CRISPR/Cas系统。之前的研究(Harrington等,Programmed DNA destruction by miniature CRISPR
‑
Cas14 enzymes,Science 362,839
‑
842(2018))首次报道了作为衍生自古细菌的CRISPR/Cas系统的CRISPR/Cas14a系统。随后的研究(Karvelis等,Nucleic Acids Research,Vol.48,No.9 5017(2020))将CRISPR/Cas14系统归类为CRISPR/Cas12f1系统。CRISPR/Cas12f1系统属于V
‑
F1系统(其为CRISPR/Cas系统的一个亚型,被归类为2 ...
【技术保护点】
【技术特征摘要】
【国外来华专利技术】1.一种用于CRISPR/Cas12f1系统的经工程化的引导RNA,包括:经工程化的支架区,和间隔区,其中,所述经工程化的支架区和所述间隔区以5
’
至3
’
的方向彼此顺序连接,所述间隔区包含10个核苷酸至50个核苷酸,并且具有与靶序列互补的序列,所述经工程化的支架区的序列不同于5
′‑
CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC
‑3′
(SEQ ID NO:7),并且所述经工程化的支架区的序列使得以下序列以5
′
至3
′
的方向彼此顺序连接:选自于由5
′‑
A
‑3′
、5
′‑
AA
‑3′
、5
′‑
GAA
‑3′
、5
′‑
AGAA
‑3′
、5
′‑
GAGAA
‑3′
、5
′‑
GGAGAA
‑3′
、5
′‑
UGGAGAA
‑3′
、5
′‑
GUGGAGAA
‑3′
、5
′‑
AGUGGAGAA
‑3′
、5
′‑
AAGUGGAGAA
‑3′
(SEQ ID NO:16)、5
′‑
AAAGUGGAGAA
‑3′
(SEQ ID NO:17)、5
′‑
UAAAGUGGAGAA
‑3′
(SEQ ID NO:18)、5
′‑
AUAAAGUGGAGAA
‑3′
(SEQ ID NO:19)、5
′‑
GAUAAAGUGGAGAA
‑3′
(SEQ ID NO:20)、5
′‑
UGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:21)、5
′‑
CUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:22)、5
′‑
ACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:23)、5
′‑
CACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:24)、5
′‑
UCACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:25)、5
′‑
UUCACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:26)和5
′‑
CUUCACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:9)所组成的组中的序列;选自于由5
′‑
CCGCUUCACUUAGAGUGAAGGUGG
‑3′
(SEQ ID NO:430)、5
′‑
CCGCUUCACCUUAGGAGUGAAGGUGG
‑3′
(SEQ ID NO:431)、5
′‑
CCGCUUCACCAUUAGUGAGUGAAGGUGG
‑3′
(SEQ ID NO:38)、5
′‑
CCGCUUCACCAAUUAGUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:39)、5
′‑
CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:40)、5
′‑
CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:41)、5
′‑
CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:42)、5
′‑
CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:43)、5
′‑
CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:44)、5
′‑
CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:45)、5
′‑
CCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:46)、5
′‑
CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:47)、5
′‑
CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:48)、5
′‑
CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:49)和5
′‑
CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:10)所组成的组中的序列;5
′‑
GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA
‑3′
(SEQ ID NO:11);选自于由5
′‑
AACAAAGAAAGGA
‑3′
(SEQ ID NO:110)、5
′‑
AACAAAUGAAAAGGA
‑3′
(SEQ ID NO:111)、5
′‑
AACAAAUUGAAAAAGGA
‑3′
(SEQ ID NO:112)、5
′‑
AACAAAUUCGAAAGAAGGA
‑3′
(SEQ ID NO:113)、5
′‑
AACAAAUUCAGAAAUGAAGGA
‑3′
(SEQ ID NO:114)、5
′‑
AACAAAUUCAUGAAAAUGAAGGA
‑3′
(SEQ ID NO:115)、5
′‑
AACAAAUUCAUUGAAAAAUGAAGGA
‑3′
(SEQ ID NO:116)和5
′‑
AACAAAUUCAUUUGAAAGAAUGAAGGA
‑3′
(SEQ ID NO:117)所组成的组中的序列;以及5
′‑
AUGCAAC
‑3′
。
2.一种用于CRISPR/Cas12f1系统的经工程化的引导RNA,包括:经工程化的支架区,和间隔区;其中,所述经工程化的支架区和所述间隔区以5
’
至3
’
的方向彼此顺序连接,所述间隔区包含10个核苷酸至50个核苷酸,并且具有与靶序列互补的序列,所述经工程化的支架区的序列不同于5
′‑
CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGAAUGCAAC
‑3′
(SEQ ID NO:315);所述经工程化的支架区的序列使得以下序列以5
′
至3
′
的方向彼此顺序连接:选自于由5
′‑
A
‑3′
、5
′‑
AA
‑3′
、5
′‑
GAA
‑3′
、5
′‑
AGAA
‑3′
、5
′‑
GAGAA
‑3′
、5
′‑
GGAGAA
‑3′
、5
′‑
UGGAGAA
‑3′
、5
′‑
GUGGAGAA
‑3′
、5
′‑
AGUGGAGAA
‑3′
、5
′‑
AAGUGGAGAA
‑3′
(SEQ ID NO:16)、5
′‑
AAAGUGGAGAA
‑3′
(SEQ ID NO:17)、5
′‑
UAAAGUGGAGAA
‑3′
(SEQ ID NO:18)、5
′‑
AUAAAGUGGAGAA
‑3′
(SEQ ID NO:19)、5
′‑
GAUAAAGUGGAGAA
‑3′
(SEQ ID NO:20)、5
′‑
UGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:21)、5
′‑
CUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:22)、5
′‑
ACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:23)、5
′‑
CACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:24)、5
′‑
UCACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:25)、5
′‑
UUCACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:26)和5
′‑
CUUCACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:9)所组成的组中的序列;选自于由5
′‑
CCGCUUCACUUAGAGUGAAGGUGG
‑3′
(SEQ ID NO:430)、5
′‑
CCGCUUCACCUUAGGAGUGAAGGUGG
‑3′
(SEQ ID NO:431)、5
′‑
CCGCUUCACCAUUAGUGAGUGAAGGUGG
‑3′
(SEQ ID NO:38)、5
′‑
CCGCUUCACCAAUUAGUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:39)、5
′‑
CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:40)、5
′‑
CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:41)、5
′‑
CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:42)、5
′‑
CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:43)、5
′‑
CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:44)、5
′‑
CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:45)、5
′‑
CCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:46)、5
′‑
CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:47)、5
′‑
CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:48)、5
′‑
CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:49)和5
′‑
CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:10)所组成的组中的序列;5
′‑
GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA
‑3′
(SEQ ID NO:11);选自于由5
′‑
AACAAAGAAAGGA
‑3′
(SEQ ID NO:110)、5
′‑
AACAAAUGAAAAGGA
‑3′
(SEQ ID NO:111)、5
′‑
AACAAAUUGAAAAAGGA
‑3′
(SEQ ID NO:112)、5
′‑
AACAAAUUCGAAAGAAGGA
‑3′
(SEQ ID NO:113)、5
′‑
AACAAAUUCAGAAAUGAAGGA
‑3′
(SEQ ID NO:114)、5
′‑
AACAAAUUCAUGAAAAUGAAGGA
‑3′
(SEQ ID NO:115)、5
′‑
AACAAAUUCAUUGAAAAAUGAAGGA
‑3′
(SEQ ID NO:116)、5
′‑
AACAAAUUCAUUUGAAAGAAUGAAGGA
‑3′
(SEQ ID NO:117)、5
′‑
AACAAAUUCAUUUUGAAACGAAUGAAGGA
‑3′
(SEQ ID NO:293)、5
′‑
AACAAAUUCAUUUUUGAAAACGAAUGAAGGA
‑3′
(SEQ ID NO:294)、5
′‑
AACAAAUUCAUUUUUCGAAAGACGAAUGAAGGA
‑3′
(SEQ ID NO:295)、5
′‑
AACAAAUUCAUUUUUCCGAAAAGACGAAUGAAGGA
‑3′
(SEQ ID NO:296)、5
′‑
AACAAAUUCAUUUUUCCUGAAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:297)、5
′‑
AACAAAUUCAUUUUUCCUCGAAAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:298)、5
′‑
AACAAAUUCAUUUUUCCUCUGAAAAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:299)、5
′‑
AACAAAUUCAUUUUUCCUCUCGAAAGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:300)、5
′‑
AACAAAUUCAUUUUUCCUCUCCGAAACGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:301)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAGAAACCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:302)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAGAAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:303)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUGAAAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:304)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUGAAAAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:305)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUCGAAAGAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:306)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUCUGAAAAGAACCCGAAUAGACGAAUGAAGGA
‑3′
(SE 0ID NO:307)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUCUGGAAACAGAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:308)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUCUGCGAAAGCAGAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:309)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUCUGCAGAAAUGCAGAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:310)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:311)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:312)、5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAGAAAUUGCAGAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:313)和5
′‑
AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAAUGAAGGA
‑3′
(SEQ ID NO:314)所组成的组中的序列;以及5
′‑
AUGCAAC
‑3′
。3.如权利要求1所述的经工程化的引导RNA,其中,所述支架区的序列使得以下序列以5
’
至3
’
的方向彼此顺序连接:5
′‑
A
‑3′
;5
′‑
CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:10);5
′‑
GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA
‑3′
(SEQ ID NO:11);5
′‑
AACAAAGAAAGGA
‑3′
(SEQ ID NO:110);以及5
′‑
AUGCAAC
‑3′
。4.如权利要求1所述的经工程化的引导RNA,其中,所述支架区的序列使得以下序列以5
’
至3
’
的方向彼此顺序连接:5
′‑
A
‑3′
;5
′‑
CCGCUUCACUUAGAGUGAAGGUGG
‑3′
(SEQ ID NO:430);5
′‑
GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA
‑3′
(SEQ ID NO:11);5
′‑
AACAAAGAAAGGA
‑3′
(SEQ ID NO:110);以及5
′‑
AUGCAAC
‑3′
。5.一种用于CRISPR/Cas12f1系统的经工程化的引导RNA,包括:
ID NO:52)和5
′‑
AAAGCUGUCCC
‑3′
(SEQ ID NO:53)所组成的组;所述第十一序列选自于由5
′‑
U
‑3′
、5
′‑
CU
‑3′
、5
′‑
ACU
‑3′
、5
′‑
AACU
‑3′
、5
′‑
GAACU
‑3′
、5
′‑
AGAACU
‑3′
、5
′‑
UAGAACU
‑3′
、5
′‑
UUAGAACU
‑3′
、5
′‑
AUUAGAACU
‑3′
、5
′‑
GAUUAGAACU
‑3′
(SEQ ID NO:54)、5
′‑
GGAUUAGAACU
‑3′
(SEQ ID NO:55)和5
′‑
GGGAUUAGAACU
‑3′
(SEQ ID NO:56)所组成的组:其中,所述第二序列的3
’
端和所述第三序列的5
’
端通过所述第十序列彼此连接;以及所述第三序列的3
’
端和所述第四序列的5
’
端通过所述第十一序列彼此连接。10.如权利要求9所述的经工程化的引导RNA,其中当第十序列是5
′‑
A
‑3′
时,第十一序列是5
′‑
U
‑3′
,当第十序列是5
′‑
AA
‑3′
时,第十一序列是5
′‑
CU
‑3′
,当第十序列是5
′‑
AAA
‑3′
时,第十一序列是5
′‑
ACU
‑3′
,当第十序列是5
′‑
AAAG
‑3′
时,第十一序列是5
′‑
AACU
‑3′
,当第十序列是5
′‑
AAAGC
‑3′
时,第十一序列是5
′‑
GAACU
‑3′
,当第十序列是5
′‑
AAAGCU
‑3′
时,第十一序列是5
′‑
AGAACU
‑3′
,当第十序列是5
′‑
AAAGCUG
‑3′
时,第十一序列是5
′‑
UAGAACU
‑3′
或5
′‑
UUAGAACU
‑3′
,当第十序列是5
′‑
AAAGCUGU
‑3′
时,第十一序列是5
′‑
AUUAGAACU
‑3′
,当第十序列是5
′‑
AAAGCUGUC
‑3′
时,第十一序列是5
′‑
GAUUAGAACU
‑3′
(SEQ ID NO:54),当第十序列是5
′‑
AAAGCUGUCC
‑3′
(SEQ ID NO:52)时,第十一序列是5
′‑
GGAUUAGAACU
‑3′
(SEQ ID NO:55),当第十序列是5
′‑
AAAGCUGUCCC
‑3′
(SEQ ID NO:53)时,第十一序列是5
′‑
GGGAUUAGAACU
‑3′
(SEQ ID NO:56),当第十序列是5
′‑
AAAAGCUGUCCC
‑3′
(SEQ ID NO:440)时,第十一序列可以是5
′‑
GGGAUUAGAACUU
‑3′
(SEQ ID NO:442),或当第十序列是5
′‑
CAAAAGCUGUCCC
‑3′
(SEQ ID NO:441)时,第十一序列是5
′‑
GGGAUUAGAACUUG
‑3′
(SEQ ID NO:443)。11.如权利要求10所述的经工程化的引导RNA,其中,所述第十序列是5
′‑
CAAAAGCUGUCCC
‑3′
(SEQ ID NO:441),并且所述第十一序列是5
′‑
GGGAUUAGAACUUG
‑3′
(SEQ ID NO:443)。12.如权利要求5所述的经工程化的引导RNA,其中,所述经工程化的支架区的序列进一步包含第十二序列和第十三序列,第十二序列选自于由5
′‑
U
‑3′
、5
′‑
UU
‑3′
、5
′‑
UUC
‑3′
、5
′‑
UUCA
‑3′
、5
′‑
UUCAU
‑3′
、5
′‑
UUCAUU
‑3′
和5
′‑
UUCAUUU
‑3′
所组成的组;第十三序列选自于由5
′‑
A
‑3′
、5
′‑
AA
‑3′
、5
′‑
GAA
‑3′
、5
′‑
UGAA
‑3′
、5
′‑
AUGAA
‑3′
、5
′‑
AAUGAA
‑3′
和5
′‑
GAAUGAA
‑3′
所组成的组,其中,所述第六序列的3
’
端和所述接头的5
’
端通过所述第十二序列彼此连接,以及所述接头的3
’
端和所述第七序列的5
’
端通过所述第十三序列彼此连接。13.如权利要求12所述的经工程化的引导RNA,其中当第十二序列是5
′‑
U
‑3′
时,第十三序列是5
′‑
A
‑3′
,当第十二序列是5
′‑
UU
‑3′
时,第十三序列是5
′‑
AA
‑3′
,
当第十二序列是5
′‑
UUC
‑3′
时,第十三序列是5
′‑
GAA
‑3′
,当第十二序列是5
′‑
UUCA
‑3′
时,第十三序列是5
′‑
UGAA
‑3′
,当第十二序列是5
′‑
UUCAU
‑3′
时,第十三序列是5
′‑
AUGAA
‑3′
,当第十二序列是5
′‑
UUCAUU
‑3′
时,第十三序列是5
′‑
AAUGAA
‑3′
,或当第十二序列是5
′‑
UUCAUUU
‑3′
时,第十三序列是5
′‑
GAAUGAA
‑3′
。14.一种用于CRISPR/Cas12f1系统的经工程化的引导RNA,包括:经工程化的支架区,和间隔区;其中,所述间隔区具有10个至50个核苷酸的长度,并且具有与靶序列互补的序列,并且经工程化的支架区的序列以5
′
至3
′
的方向包括:经工程化的tracrRNA,其中,以下的序列彼此连接:选自于由5
′‑
A
‑3′
、5
′‑
AA
‑3′
、5
′‑
GAA
‑3′
、5
′‑
AGAA
‑3′
、5
′‑
GAGAA
‑3′
、5
′‑
GGAGAA
‑3′
、5
′‑
UGGAGAA
‑3′
、5
′‑
GUGGAGAA
‑3′
、5
′‑
AGUGGAGAA
‑3′
、5
′‑
AAGUGGAGAA
‑3′
(SEQ ID NO:16)、5
′‑
AAAGUGGAGAA
‑3′
(SEQ ID NO:17)、5
′‑
UAAAGUGGAGAA
‑3′
(SEQ ID NO:18)、5
′‑
AUAAAGUGGAGAA
‑3′
(SEQ ID NO:19)、5
′‑
GAUAAAGUGGAGAA
‑3′
(SEQ ID NO:20)、5
′‑
UGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:21)、5
′‑
CUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:22)、5
′‑
ACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:23)、5
′‑
CACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:24)、5
′‑
UCACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:25)、5
′‑
UUCACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:26)和5
′‑
CUUCACUGAUAAAGUGGAGAA
‑3′
(SEQ ID NO:9)所组成的组中的序列;选自于由5
’‑
CCGCUUCACUUAGAGUGAAGGUGG
‑3’
(SEQ ID NO:ID NO:430)、5
′‑
CCGCUUCACCUUAGGAGUGAAGGUGG
‑3′
(SEQ ID NO:431)、5
′‑
CCGCUUCACCAUUAGUGAGUGAAGGUGG
‑3′
(SEQ ID NO:38)、5
′‑
CCGCUUCACCAAUUAGUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:39)、5
′‑
CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:40)、5
′‑
CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:41)、5
′‑
CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:42)、5
′‑
CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:43)、5
′‑
CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:44)、5
′‑
CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:45)、5
′‑
CCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:46)、5
′‑
CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:47)、5
′‑
CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:48)、5
′‑
CCGCUUCACCAAAAGCUGUCCUUAGGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:49)和5
′‑
CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGG
‑3′
(SEQ ID NO:10)所组成的组中的序列;5
′‑
GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA
‑3′
(SEQ ID NO:1 1);以及选自于由5
′‑
AACAAA
‑3′
、5
′‑
AACAAAU
‑3′
、5
′‑
AACAAAUU
‑3′
、5
′‑
AACAAAUUC
‑3′
、5
′‑
AACAAAUUCA
‑3′
(SEQ ID NO:66)、5
′‑
AACAAAUUCAU
‑3′
(SEQ ID NO:67)、5
′‑
AACAAAUUCAUU
‑3′
(SEQ ID NO:68)和5
′‑
AACAAAUUCAUUU
‑3′
(SEQ ID NO:12)所组成的组中的序列;经工程化的crRNA重复序列部分,其中以下的序列彼此连接:选自于由5
′‑
GGA
‑3′
、5
′‑
AGGA
‑3′
、5
′‑
AAGGA
‑3′
、5
′‑
GAAGGA
‑3′
、5
′‑
UGAAGGA
‑3′
、5
′‑
AUGAAGGA
‑3′
、5
′‑
AAUGAAGGA
‑3′
和5
′‑
GAAUGAAGGA
‑3′
(SEQ ID NO:14)所组成的组中的序列;以及5
′‑
AUGCAAC
‑3′
;其中,所述经工程化的crRNA重复序列的3
’
端与所述间隔区的5
’
端连接,并且其中,将如下排除:其中的经工程化的tracrRNA的序列与5
′‑
CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU
‑3′
(SEQ ID NO:1)相同,并且经工程化的crRNA重复序列与5
′‑
GAAUGAAGGAAUGCAAC
‑3′
(SEQ ID NO:3)相同。15.经工程化的CRISPR/Cas12f1复合物,包含:Cas12f1蛋白;和经工程化的引导RNA,其中,所述经工程化的引导RNA包括:经工程化的支架区;和间隔区;其中,所述经工程化的支架区和所述间隔区以5
’
至3
’
的方向彼此顺序连接,所述间隔区包括10个核苷酸至50个核苷酸,并且具有与靶序列互补的序列,所述经工程化的支架区的序列不同于5
′‑
CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC
‑3′
(SEQ ID NO:7),并且所述经工程化的支架区的序列使得以下序列以5
’
至3
’
的方向彼此顺序连接:选自于由5
′‑
A
‑3′
、5
′‑
AA
‑3′
、5
′‑
GAA
‑3′
、5
′‑
AGAA
‑3′
、5
′‑
GAGAA
‑3′
、5
′‑
GGAGAA
‑3′
、5
′‑
UGGAGAA
‑3′
...
【专利技术属性】
技术研发人员:金龙三,金掉妍,
申请(专利权)人:基恩科雷有限责任公司,
类型:发明
国别省市:
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