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。
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.
作者
高伟欣
黄火清
赵晶
张鑫
杨宁
杨浩萌
GAO Wei-xin;HUANG Huo-qing;ZHAO Jing;ZHANG Xin;YANG Ning;YANG Hao-meng(Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193)
出处
《生物技术通报》
CAS
CSCD
北大核心
2022年第8期60-68,共9页
Biotechnology Bulletin
基金
国家重点研发计划(2021YFC2100204)。
