基于芯片DNA合成和单细胞拉曼表型分选的基因体内功能筛选新方法研究

Screening of in vivo gene function has been one key approach for discovering new drugs, enzymes and cells, yet its power is dependent on the high-throughput and cost of gene synthesis and on techniques that phenotype the host cells carrying the gene. This project proposes to develop an integrated method for in vivo gene function screening by simultaneously taking advantage of on-chip DNA synthesis which is of high-throughput and low-cost and single-cell Raman spectroscopy in phenotype of cells does not require labeling and culture. Fatty acyl-ACP thioesterase will be employed as a model to develop and demonstrate this novel method. By optimizing and coupling high-throughput DNA synthesis, single-cell Raman analysis for distinguishing content, side-chain length and unsatiation degree of fatty acids in the cell and Raman-activated single-cell sorting and sequencing, we will identify novel enzyme gene mutants from a random mutagenesis library of enzyme genes, so as to unveil the sequence-function relationship of the enzyme gene. In the end, a novel in vivo gene function screening method that is low-cost, high-throughput, label-free, culture-independent and screening at a resolution of a single cell will be established and validated. These efforts should significantly expand the application of in vivo gene function in many areas and also shed new light on the sequence-function relationship of fatty acyl-ACP thioesterases.

基因体内功能(in vivo function)筛选是发现新酶的重要途径，但其应用受限于基因合成的通量与成本，以及相应的细胞表型筛选技术。本研究同时利用和耦合芯片合成基因突变体库低成本、高通量的特色和单细胞拉曼表型筛选技术无需标记、无需细胞培养的优势，以脂肪酰ACP硫酯酶(fatty acyl-ACP thioesterase)为模型，通过优化与耦合芯片DNA高通量合成、单细胞拉曼检测区分脂肪酸的含量、链的长度及饱和度以及与拉曼检测耦合的单细胞分选和测序等关键技术环节，筛选出新的脂肪酰ACP硫酯酶突变体，从而研究该基因上功能位点与产物含量、侧链的长度及饱和度的关联机制，最终建立和示范一种低成本、高通量、不须目标产物标记、不再依赖于培养、以单个细胞为筛选单位的新型基因体内功能筛选技术。这些努力将有力拓展基因体内功能筛选技术在诸多领域的应用，同时为脂肪酰ACP硫酯酶的构效机制提供新的理解。

来源于不同物种的同源基因的合成是发现新的基因功能的重要途径，但其应用受限于基因合成的通量、保真性与成本。本研究同时利用和耦合芯片合成基因库低成本、高通量的特色和基于MutS蛋白的错配纠正系统的优势，以来源于不同物种的Cas9同源基因为模型，通过优化与耦合芯片DNA高通量合成、MutS蛋白的基因纠错等关键技术环节，筛选出新的有功能的Cas9同源基因，从而研究该Cas9同源基因形式功能的关联机制，最终建立和示范一种低成本、高通量、高保真性的新型基因的发现和筛选技术。这些努力将有力拓展新型基因的发现及其在诸多领域的应用，同时为Cas9同源基因的构效机制提供新的理解。