质粒
遗传学
基因
大肠杆菌
基础(拓扑)
生物
内含子
碱基对
计算生物学
数学
数学分析
作者
Guzhen Cui,Dengxiong Hua,Xingxing Zhao,Jia Zhou,Ying Yang,Ting‐Yu Huang,Xin Wang,Yan Zhao,Ting Zhang,Jian Liao,Zhizhong Guan,Peng Luo,Zhenghong Chen,Xiaolan Qi,Wei Hong
标识
DOI:10.1128/spectrum.03159-22
摘要
Thermophilic group II intron is one type of retrotransposon composed of intron RNA and intron-encoded protein (IEP), which can be utilized in gene targeting by harnessing their novel ribozyme-based DNA integration mechanism termed "retrohoming." It is mediated by a ribonucleoprotein (RNP) complex that contains the excised intron lariat RNA and an IEP with reverse transcriptase (RT) activity. The RNP recognizes targeting sites by exon-binding sequences 2 (EBS2)/intron-binding sequences 2 (IBS2), EBS1/IBS1, and EBS3/IBS3 bases pairing. Previously, we developed the TeI3c/4c intron as a thermophilic gene targeting system-Thermotargetron (TMT). However, we found that the targeting efficiency of TMT varies significantly at different targeting sites, which leads to a relatively low success rate. To further improve the success rate and gene-targeting efficiency of TMT, we constructed a Random Gene-targeting Plasmids Pool (RGPP) to analyze the sequence recognition preference of TMT. A new base pairing, located at the -8 site between EBS2/IBS2 and EBS1/IBS1 (named EBS2b-IBS2b), increased the success rate (2.45- to 5.07-fold) and significantly improved gene-targeting efficiency of TMT. A computer algorithm (TMT 1.0), based on the newly discovered sequence recognition roles, was also developed to facilitate the design of TMT gene-targeting primers. The present work could essentially expand the practicalities of TMT in the genome engineering of heat-tolerance mesophilic and thermophilic bacteria.
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