优化sgRNA提高塔宾曲霉基因编辑效率  

Improving the Efficiency of Gene Editing by Optimizing sgRNA in Aspergillus tubingensis

作　　者：梁丽存 王克芬 宋祖洹 刘梦婷 李佳玉 罗会颖 姚斌 杨浩萌 LIANG Li-cun;WANG Ke-fen;SONG Zu-huan;LIU Meng-ting;LI Jia-yu;LUO Hui-ying;YAO Bin;YANG Hao-meng(Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193;Longkete Enzyme Preparations,Linyi 276000)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]山东隆科特酶制剂有限公司,临沂276000

出　　处：《生物技术通报》2025年第3期62-70,共9页Biotechnology Bulletin

基　　金：国家重点研发计划(2021YFC2100204);国家现代农业产业技术体系专项(CARS-41)。

摘　　要：【目的】在CRISPR/Cas9基因编辑系统中,sgRNA是重要的基因编辑元件之一,它与Cas9蛋白结合并通过间隔区序列与基因组DNA互补,引导Cas9蛋白对基因组进行精准剪切。为了提高塔宾曲霉的基因编辑效率,对sgRNA进行优化是一项可行的策略。【方法】对sgRNA的启动子和发夹结构进行了优化,并在塔宾曲霉中进行了基因编辑效率的验证。【结果】通过RNP法进行基因编辑时,带有“lock”结构的sgRNA的基因编辑效率比对照组提高了9.37%;sgRNA在进行体内表达时,使用tRNA^(Gly15)和tRNA^(Gly17)启动子的基因编辑效率分别高出5S rRNA启动子14%-16%;使用tRNA^(Gly15)启动子表达带有“lock”结构的sgRNA,能提高白孢率,并使塔宾曲霉的基因编辑效率提高到96%。【结论】“lock”结构和两种tRNA^(Gly15)和tRNA ^(Gly17)启动子能够提高塔宾曲霉的基因编辑效率。【Objective】In the CRISPR/Cas9 gene editing system,sgRNA is one of the important gene editing elements.It binds to the Cas9 protein and complements the genomic DNA through the spacer sequence to guide the Cas9 protein to accurately cut the genome.In order to improve the gene editing efficiency of Aspergillus tubingensis,sgRNA optimization is a feasible strategy.【Method】The promoter and hairpin structure of sgRNA were optimized,and the gene editing efficiency was verified in A.tubingensis.【Result】The gene editing efficiency of sgRNA with“lock”structure was 9.37%higher than that of the control group when gene editing was performed by RNP method.When sgRNA was expressed in vivo,the gene editing efficiency of tRNA^(Gly15) and tRNA^(Gly17) promoters was 14%-16%higher than that of 5S rRNA promoter,respectively.Using the tRNA^(Gly15) promoter to express sgRNA with a“lock”structure increased the white spore rate and increased the gene editing efficiency of A.tubingensis to 96%.【Conclusion】The“lock”structure and two promotors of tRNA^(Gly15) and tRNA^(Gly17) may improve the gene editing efficiency of A.tubingensis.

关 键 词：塔宾曲霉 基因编辑效率 启动子 “lock”结构 

分 类 号：Q78[生物学—分子生物学]