TeI3c/4c嗜热二型内含子单插入热点碱基组成规律及其应用研究

Thermotargetron(TMT) is a gene targeting system derived from thermophilic mobile group II introns(TeI3c/4c). It has a broad host range, with the ability to function in a wide temperature range and can be used in bacteria with low transformation efficiency. Thus, it has the potential to be used for gene targeting in a variety of thermophiles, as well as mesophiles that can tolerate short periods at elevated temperatures. However, low targeting (28%) and high off-target rate(50%) is the critical technical bottleneck that preventing TMT from widely application. In our previous study, we found that poorly designed re-targeting sites, which caused by unclear of the nucleotides composition law at the single insertion sites, were the main cause of low targeting and high off-target rate. In order to solve this problem, Firstly, we will perform multi-locus gene inactivation experiment in Escherichia coli model to acquire gene inactivation mutants. Afterward, establish a single insertion database using the sequence at the single insertion sites, which determined by Southern blot and sequencing techniques. Secondly, analyze the nucleotides composition laws of single insertion sites within the database, and the results will be used to train a computer algorithm to improve TMT`s targeting efficiency while reducing the off-target rate in mosophilic and thermophilic Clostridia. Finally, a new gene inactivation system of high efficiency and preciseness, which can be applied universally in mosophilic and thermophilic Clostridia, will be obtained.

基于嗜热二型内含子TeI3c/4c逆转录归巢效应构建的靶向基因失活系统Thermotargetron (TMT)，因其宿主范围广、热稳定性好、不依赖较高转化效率等优点，具有在高温及能短暂耐受高温的中温梭菌中应用的潜力。然而该系统目前打靶成功率低(28%)且脱靶率高(50%)，是阻碍TMT广泛应用的关键技术瓶颈。我们前期研究发现，因TMT核心元件TeI3c/4c单插入热点碱基组成规律不明确导致的靶位点设计不合理，是造成打靶成功率低和脱靶的主要原因。为解决该问题，本项目首先在已建立的嗜热二型内含子大肠杆菌基因打靶模型中，实施多基因靶向失活实验，并借助核酸杂交和测序技术，筛选和测定单插入位点序列组成，从而建立TMT单插入热点数据库；然后，利用该数据库分析TMT单插入热点序列规律，并指导靶位点评价软件的开发，在提高其打靶效率的同时减少脱靶的产生。最终获得一套通用、高效、严谨的梭菌靶向基因失活系统。

本项目的目标是研究Thermotargetron（TMT）高频和单插入热点碱基组成规律，提高TMT在不同应用环境（如中温微生物和高温微生物）打靶成功率和降低其脱靶效率。通过本项目的开展，共获得了42例成功和74例失败的打靶位点碱基序列。基于对以上序列碱基组成规律的分析，发现TMT高效打靶位点识别规律为：识别序列+5和+6位为‘A’或‘T’时打靶成功率高；识别序列+12位为‘T’时打靶失败率高；识别位点+7位的‘C’和+10位的‘G’或‘T’可以降低TMT打靶效率。基于该规律，我们开发了一套自动寻找TMT高效打靶位点和引物设计程序（Thermotargetron finder v2.0）。不仅如此，我们还应用该系统在大肠杆菌和艰难梭菌中实现了高效、严谨基因失活（直接控制TMT在中温菌中的打靶时间）。然而，遗憾的是当Thermotargetron finder v2.0软件应用于热纤梭菌基因失活时，基因打靶成功率虽有提高，但是脱靶率却没有改善。综上，通过本研究的开展建立了一套温度适应范围广、高效的靶向基因失活系统，该系统为中温及高温微生物基因功能研究提供了新的思路和技术。