基于CRISPR/Cpf1系统下的诱导型多基因激活体系的建立

An inducible CRISPR/Cpf1 system for multiplex gene activation

摘要:

为开发高效率的内源多基因激活工具，利用CRISPR/Cpf1系统能够促使precrRNA中多个crRNA自我剪切和释放的特性，将As/Fn/LbCpf1的核酸酶区域定点突变形成dCpf1，并在C端和N端分别引入了VPR三元转录激活域和不稳定结构域（DD-domain）、四环素控制的操纵子，构建了TRE3G-DD-dCpf1-VPR激活系统.结果显示，Oct4基因的转录水平提升高达104倍，且Myod1、Oct4基因的转录水平分别在Dox和TMP单药的控制下，能够实现不同程度的转录水平的提升，在加双药的条件下Myod1、Oct4基因可分别提高80、120多倍.证明该系统能够通过靶向DNA快捷、高效的进行诱导内源性多基因激活.

Abstract:

In order to develop highefficiency endogenous multigene activation tools, and based on the CRISPR/Cpf1 system, which can promote the self-cleavage and release of multiple crRNAs in pre-crRNA, the TRE3G-DD-dCpf1-VPR activation system was constructed. In this system, the sitedirected mutation of the nuclease region of As/Fn/LbCpf1 to form dCpf1, and The VPR with DD-domain the tetracycline-controlled operon were introduced at the C and N terminal, respectively. The results showed that the transcription level of Oct4 gene was increased by 104 times, and the transcription levels of Myod1 and Oct4 genes can achieve different levels under the control of Dox and TMP single drugs respectively. Under the condition of adding dual drugs, Myod1 and Oct4 genes can be increased by more than 80 and 120 times respectively. It proves that the system can induce endogenous multi-gene activation quickly and efficiently by targeting DNA.