应用于基因编辑的核糖核蛋白复合体的构建与活性验证

doi:10.13560/j.cnki.biotech.bull.1985.2021-1353

摘要/Abstract

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。

关键词: Cas9蛋白, RNP复合体, 大肠杆菌, 活性验证

Abstract:

CRISPR/Cas9 gene editing technology has the advantages of simple operation,low cost and high editing efficiency,and has been widely used in the research of gene editing in animals,plants and microorganisms. In this technical system,ribonucleoprotein（RNP）complex assembled from Cas9 protein and guide RNA（gRNA）,exerts the function of nucleic acid cleavage. At present,there are more and more researches on direct delivery of RNP complex assembled in vitro into recipient cells for gene editing,which has become an effective means to improve editing efficiency,and reduce off-target rate. The purpose of this study is to obtain an RNP complex with high cleavage activity. In this research, the Cas9 protein was successfully expressed in Escherichia coli BL21（DE3）,and the recombinant Cas9 protein was purified by His-tag affinity resin. At the same time,the T7 transcription kit in vitro transcription and purification of gRNA was performed,so that the purified Cas9 protein and gRNA spontaneously assembled into RNP complexes. After in vitro activity detection,RNP complexes demonstrated good DNA double-stranded shearing activity;and RNP was co-transformed the Aspergillus niger strain with donor DNA,the target gene was successfully knocked out,and the editing efficiency reached more than 90%. The RNP complex obtained in this study can be applied to gene editing of filamentous fungi,saving time and cost,and promoting the application of RNP-mediated gene editing technology in more fields.

Key words: Cas9 protein, RNP complex, Escherichia coli, activity detection

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.

图/表 8

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

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

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3 RNP复合体体内编辑过程示意图

Fig.3 Schematic diagram of RNP complex editing process in vivo

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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