生物技术通报 ›› 2022, Vol. 38 ›› Issue (8): 60-68.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1353
收稿日期:
2021-10-29
出版日期:
2022-08-26
发布日期:
2022-09-14
作者简介:
高伟欣,女,硕士研究生,研究方向:丝状真菌蛋白表达系统;E-mail: 基金资助:
GAO Wei-xin(), HUANG Huo-qing, ZHAO Jing, ZHANG Xin, YANG Ning, YANG Hao-meng()
Received:
2021-10-29
Published:
2022-08-26
Online:
2022-09-14
摘要:
CRISPR/Cas9基因编辑技术具有操作简单、成本低廉和编辑效率高等优点,已经被广泛应用于动物、植物、微生物的基因编辑研究。在该技术体系中,发挥核酸剪切功能的是Cas9蛋白和guideRNA(gRNA)组装而成的核糖核蛋白复合体,即RNP(ribonucleoprotein)复合体。目前,应用体外组装的RNP复合体直接递送进入受体细胞进行基因编辑的研究越来越多,成为提高编辑效率、降低脱靶率的有效手段。本研究以获得具有高效剪切活性的RNP复合体为试验目的,在大肠杆菌BL21(DE3)中成功表达了Cas9蛋白,并通过His-tag亲和树脂对重组Cas9蛋白进行了纯化;同时,通过T7转录试剂盒对gRNA进行体外转录和纯化,使纯化的Cas9蛋白和gRNA自发组装成RNP复合体,经体外活性检测,RNP复合体具有很好的DNA双链剪切活性;将其与donor DNA共同转化到黑曲霉菌株,成功敲除了目标蛋白,编辑效率达到90%以上。本研究获得的RNP复合体,可以应用于丝状真菌的基因编辑,节约了试验时间与成本,并推动RNP介导的基因编辑技术在更多领域的应用。
高伟欣, 黄火清, 赵晶, 张鑫, 杨宁, 杨浩萌. 应用于基因编辑的核糖核蛋白复合体的构建与活性验证[J]. 生物技术通报, 2022, 38(8): 60-68.
GAO Wei-xin, HUANG Huo-qing, ZHAO Jing, ZHANG Xin, YANG Ning, YANG Hao-meng. Construction and Activity Verification of Ribonucleoprotein Complex for Gene Editing[J]. Biotechnology Bulletin, 2022, 38(8): 60-68.
Primer name | Primer sequence(5'-3') |
---|---|
Pet-Insert-F | taagaaggagatataccatggATAAGAAATACTCAATAGGCTTAGA |
Pet-Insert-R | gtggtggtggtggtgctcgagGACCTTGCGCTTCTTCTTGGgagggtcacctcctagctgactcaaatc |
glucoamylase-F | atgtcgttccgatctctactcgc |
glucoamylase-R | ctaccgccaggtgtcagtcac |
223up-F | atgtcgttccgatctctactcgccctg |
223up-R | acttcttcccagagatctgcgatcg |
223down-F | caatggctcgtctttctttacgattgc |
223down-R | aggtgacagtcacactgctgtagg |
hph-F | gcagatctctgggaagaagtgacagaagatgatattgaaggagcac |
hph-R | aaagaaagacgagccattggtatctggaagaggtaaacccgaaacg |
hph-jc-F | ctcgccgatagtggaaaccgac |
hph-jc-R | cgcaccaagttatcgtgcacc |
GA223homo-up-jc-F | cccacaccatcggagattcgtcgcc |
表1 引物名称及序列信息
Table 1 Primer names and sequence information
Primer name | Primer sequence(5'-3') |
---|---|
Pet-Insert-F | taagaaggagatataccatggATAAGAAATACTCAATAGGCTTAGA |
Pet-Insert-R | gtggtggtggtggtgctcgagGACCTTGCGCTTCTTCTTGGgagggtcacctcctagctgactcaaatc |
glucoamylase-F | atgtcgttccgatctctactcgc |
glucoamylase-R | ctaccgccaggtgtcagtcac |
223up-F | atgtcgttccgatctctactcgccctg |
223up-R | acttcttcccagagatctgcgatcg |
223down-F | caatggctcgtctttctttacgattgc |
223down-R | aggtgacagtcacactgctgtagg |
hph-F | gcagatctctgggaagaagtgacagaagatgatattgaaggagcac |
hph-R | aaagaaagacgagccattggtatctggaagaggtaaacccgaaacg |
hph-jc-F | ctcgccgatagtggaaaccgac |
hph-jc-R | cgcaccaagttatcgtgcacc |
GA223homo-up-jc-F | cccacaccatcggagattcgtcgcc |
Name | Sequence(5'-3') |
---|---|
gRNA | TAATACGACTCACTATAGGgatctctgggaagaagtcaa GTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT |
cgRNA | TAATACGACTCACTATAGGgatgggattcgtgagtacaaGTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT |
表2 RNA体外转录模板的序列信息
Table 2 Sequence information of in vitro transcription templates of RNA
Name | Sequence(5'-3') |
---|---|
gRNA | TAATACGACTCACTATAGGgatctctgggaagaagtcaa GTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT |
cgRNA | TAATACGACTCACTATAGGgatgggattcgtgagtacaaGTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT |
添加量Additive amount | ||||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | |
Cas9 | 200 ng | 400 ng | 800 ng | 1.6 μg | 3.2 μg | 6.4 μg |
gRNA | 200 ng | 400 ng | 800 ng | 1.6 μg | 3.2 μg | 6.4 μg |
DNA | 200 ng | 200 ng | 200 ng | 200 ng | 200 ng | 200 ng |
表3 RNP复合体活性验证试验组反应体系
Table 3 Reaction system of test group for RNP complex activity verification
添加量Additive amount | ||||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | 5 | 6 | |
Cas9 | 200 ng | 400 ng | 800 ng | 1.6 μg | 3.2 μg | 6.4 μg |
gRNA | 200 ng | 400 ng | 800 ng | 1.6 μg | 3.2 μg | 6.4 μg |
DNA | 200 ng | 200 ng | 200 ng | 200 ng | 200 ng | 200 ng |
图1 重组Cas9蛋白的SDS-PAGE分析 M:蛋白分子量标准;1:诱导后菌体破碎上清;2:纯化后的Cas9蛋白
Fig.1 SDA-PAGE analysis of recombinant Cas9 M:Protein molecular mass marker;1:disrupted cell supernatant after induction;2:purified Cas9
图2 RNP复合体体外活性验证 A:阴性对照组;B:试验组,1-6(①-⑥)分别表示cCas9(Cas9)、gRNA、DNA不同浓度的添加量,三者添加量如表3所示;M:DNA分子量标准
Fig.2 In vitro activity verification of RNP complex A: Negative control. B: Experimental group, 1-6(①-⑥)respectively indicates the addition amount of cCas9(Cas9),gRNA,and DNA at different concentrations. The addition amount of the three is shown in Table 3. M:DNA molecular mass marker
图4 阳性转化子的PCR验证 M:DNA分子量标准;1-11为不同阳性转化子;CK为原始菌株对照
Fig.4 Verification of positive transformants by PCR M:DNA molecular mass marker;1-11 are different positive transformants;CK is the original strain as control
图5 转化子发酵液上清的SDS-PAGE分析 M:蛋白分子量标准;1-11为不同转化子发酵液上清;CK为原始菌株对照
Fig.5 SDS-PAGE analysis of supernatant of transformant fermentation broth M:Protein molecular mass marker;1-11 are the fermentation broth supernatants of different transformants;CK is the original strain as control
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