CRISPR/Cas9介导的基因组进化构建固态发酵耐热酵母及机理研究

项目名称： CRISPR/Cas9介导的基因组进化构建固态发酵耐热酵母及机理研究

项目编号： No.31600067

项目类型： 青年科学基金项目

立项/批准年度： 2017

项目学科： 生物科学

项目作者： 李鹏松

作者单位： 清华大学

项目金额： 17万元

中文摘要： 热胁迫是酵母固态发酵产乙醇过程中的重要限制因素。酵母的耐热性属于复杂表型，涉及到多个基因的协同作用，这类复杂表型往往由于机理模糊而难以对其进行改造。本项目拟将基因组编辑技术CRISPR/Cas9与定向进化策略相整合，建立一种CRISPR/Cas9介导的基因组进化（CRISPR/Cas9-assisted genome evolution, CAGE）新方法。首先利用转录组测序及生物信息学分析，确定热胁迫条件下酵母的差异表达基因；再通过分别构建以下调表达基因和上调表达基因为靶基因的sgRNA文库，进行逐轮迭代CRISPR/Cas9筛选，逐步提高酵母的耐热性能；最后，通过综合分析转录组测序结果和迭代CRSIPR/Cas9筛选所确定的关键基因，阐明酵母耐热表型的分子调控机制。本项目的实施将为微生物复杂表型的定向改造及机理解释提供新的思路。

中文关键词： 合成生物学育种；定向改造；CRISPR/Cas9；基因组进化；耐热酵母

英文摘要： Thermal stress is an important limiting factor for solid-state fermentation with yeast. Thermotolerance of yeast is a complex phenotype which involves synergistic actions of many genes. Such complex phenotypes are often difficult to engineer due to their unclear mechanisms. The proposed project is designed to construct a novel method named as CRISPR/Cas9-assisted genome evolution (CAGE), which combines the genome-editing tool CRISPR/Cas9 and the strategy of directed evolution. Firstly, the genes with differential expressions under thermal stress are to be identified by transcriptome sequencing and bioinformatic analysis. Secondly, by respectively constructing sgRNA libraries recognizing down-regulated and up-regulated genes followed by iterative CRISPR/Cas9 screening, the thermotolerance of yeast is to be improved step by step. Finally, by comprehensively analyzing the results of transcriptome sequencing and the key genes identified by iterative CRISPR/Cas9 screening, the molecular regulation mechanism of thermotolerance in yeast is to be clarified. The implementation of the proposed project will provide a new idea for directional modification and mechanism explanation of complex phenotypes in microorganisms.

英文关键词： Synthetic Biology Breeding;Directional Modification;CRISPR/Cas9;Genome Evolution;Thermotolerant Yeast