随着在基因定位、克隆、基因诊断等方面的突破，基因治疗有望从源头上根治神经系统遗传病。目前，CRISPR/Cas9系统在大片段DNA插入治疗上还存在不足。申请者基于前期piggyBac（PB）转座系统的研究基础，推测该系统可以有效弥补CRISPR/Cas9的上述不足。拟开展以下研究：1）根据PB转座酶（PBase）的关键氨基酸位点将其分割成12对截断体，然后结合FKBP/FRB系统对12对截断体进行活性筛选，从中筛选出活性最高的截断体组合；2）将筛选出来的PBaseN端和C端分别与dCas9（dead Cas9）融合，利用CRISPR/dCas9的高效DNA靶向性实现PB的定点转座；进而结合光遗传学系统实现光诱导的定点转座。3）运用上述技术探索在体基因组编辑对遗传性焦虑症小鼠模型的治疗作用。通过实施本项目将开发一种定点、高效、可控的PB转座系统，为神经精神疾病治疗提供新的思路和技术手段。

Nervous system genetic disorders have familial and permanent characteristic, causing great threat to human health. As the breakthrough of gene location, diagnosis and cloning technique of nervous system genetic disorders, gene therapy is becoming a novel approach to treat diseases by introducing functional genes into the genome or correcting some mutation loci. Currently, CRISPR/Cas9 system is one of the most effective method for gene therapy, but it also exhibits some insufficiency in gene insertional therapy, especially in large DNA fragment insertion. Based on our previous study, piggyBac (PB) transposition system has the potential to overcome this deficiency. By combining the advantages of CRISPR/Cas9 and PB systems, the present study aims to truncate PB transposase (PBase) into two parts (N terminus and C terminus) according to the key amino acids distribution in the protein sequence. We will select the best combination according to activity screening of twelve pairs of the truncated PBase using the FKBP/FRB system, followed by fusing with the dCas9 (dead Cas9) to achieve site-specific transposition. Furthermore, the optogenetics system will be used based on the above constructed platform to set up a light-inducible transposition system. Based on the above researches, we aim to treat genetically anxiety mice in vivo using the new genome editing platform which we established. The present project will develop a site-specific, high-efficiency, and controllable PB transposition system, which can overcome technical deficiencies of DNA insertional therapy and also provide a new pharmacological strategy for the future gene therapy.