基于核酶的自剪切sgRNA及其在基因编辑中的应用

Ribozymes based self-cleavage sgRNA and its application in gene editing

摘要:

为减少在CRISPR/Cas9基因编缉系统中,sgRNA通常由Ⅲ型启动子持续转录产生,Ⅲ型启动子不易控制,sgRNA持续表达又容易产生细胞毒性这一缺陷,本文拟建立由Ⅱ型启动子转录,并由内含子中释放sgRNA的方法.我们在sgRNA的两端设计了三种“核酶sgRNA核酶”的组合,通过核酶的自剪切作用实现sgRNA的释放.实验结果表明,HHRzsgRNAHDVRz的设计能够正确的从内含子中释放sgRNA,并且该sgRNA在构建的copGFP敲除模型中能够实现有效的基因编辑.这一发现证明在哺乳动物细胞中利用Ⅱ型启动子,在核酶和内含子剪切的协助下可以实现sgRNA的表达,这为Cas9和sgRNA整合在一个Ⅱ型启动子之下实现组织特异性和药物诱导性表达提供了基础.

Abstract:

Currently, sgRNAs are usually transcribed by type Ⅲ promoters in CRISPR/Cas9 system, which makes it difficult to achieve tissuespecific and timespecific expression. Cytotoxicity is an additional concern for constitutive transcription of small RNAs. In this paper, we designed an intron-based approach for sgRNA expression by type Ⅱ promoters, which uses ribozyme switches to facilitate sgRNA release from the spliced intron. We designed three combinations of "ribozymesgRNAribozyme" at both ends of sgRNAs to release them through the self-cleavage of ribozymes. The results indicated that the design of HHRz-sgRNA-HDVRz could correctly release sgRNA from the intron, and the released sgRNAs could achieve gene editing in human cells. Our finding demonstrated that an intron-based sgRNA expression cassette was compatible with type Ⅱ promoters and ribozyme switches could be used to facilitate sgRNAs release for gene editing.