利用单转录本表达Cas9和sgRNA条件性编辑果蝇基因组

Conditional editing of the Drosophila melanogaster genome using single transcripts expressing Cas9 and sgRNA

CRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9)系统具有高效、简单、易编辑等特性,在基因工程方面具有巨大潜能。在果蝇(Drosophila melanogaster)基因功能研究中,CRISPR/Cas9系统也得到了广泛应用。然而利用传统的CRISPR/Cas9系统编辑果蝇基因时,Cas9与sgRNA表达元件往往分别存在于不同果蝇中,需要通过复杂的遗传杂交过程将Cas9与sgRNA整合到一个个体中,操作周期长且较为复杂。本研究在CRISPR/Cas9系统基础上引入tRNA-sgRNA系统和triplex元件,利用triplex元件连接Cas9和tRNA-sgRNA基因,稳定单转录本切割后Cas9 mRNA的末端,实现一个转录本可以同时表达Cas9蛋白和sgRNA,通过一次杂交即可得到相应表型的后代,简化了遗传操作过程。本研究利用这一新的条件性基因编辑系统,分别对果蝇眼部white(w)基因和翅成虫盘broad(br)基因进行条件性敲除,观察到与预期相符的对应表型。因此,该条件性基因编辑系统在高效性、可扩展性和易操作性等方面是对现有CRISPR/Cas9系统进行的重大改进。

The CRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPRassociated protein 9)system,a highly efficient,simple,and easy genome editing technology,offers significant potential for genetic engineering and has been commonly applied in gene function studies in Drosophila melanogaster.However,when using CRISPR/Cas9 system to edit Drosophila melanogaster gene,Cas9 and sgRNA expression elements exist in different Drosophila melanogaster individuals,and Cas9 and sgRNA must be integrated into an individual through a complex genetic hybridization process,which has a long and complex operation cycle In this study,on the basis of the CRISPR/Cas9 system,we introduced the tRNA-sgRNA system and triplex elements,used triplex elements to link Cas9 and tRNA-sgRNA genes,stabilized the end of Cas9 mRNA after single transcript cutting,and made the expression of both Cas9 protein and sgRNA with a single transcript a reality.And as we obtained the corresponding phenotypic progeny in one hybridization,genetic manipulation was simplified.We found that conditional knockout of the white(w)gene in the Drosophila melanogaster eye and the broad(br)gene in the adult wing disc resulted in corresponding phenotypes that matched expectations using our new conditional gene editing system.So the significant advances in this new conditional gene editing system over the existing CRISPR/Cas9 system are that it is more efficient,extendable,and easy to use.