基因编辑技术及其在糖生物学研究中的应用

The Gene Editing Technology and Their Application in Glycobiology

基因功能的解析是现代生物学研究的基本问题,基因的精准、高效和靶向编辑是基因功能研究不可或缺的手段。在过去的30多年,基因编辑实现了从基于同源重组修复技术的基因打靶技术(gene targeting)到基于锌指核酸酶、转录激活样效应因子核酸酶和CRISPR相关核酸酶等可编程核酸酶的可控编辑技术的革命性转变。这些技术的发展极大地促进了基因功能的研究并促生了疾病治疗的颠覆性技术。本文综述了CRISPR-Cas的分类、组成及其在细菌中发挥免疫防御作用的工作原理,重点综述了基于CRISPR-Cas系统的最新基因编辑工具包括基因转录编辑器、表观遗传编辑器、碱基编辑器、先导编辑器以及靶向RNA的RCas编辑系统。随后综述了基因编辑器的细胞和器官靶向递送策略,主要讨论了腺相关病毒、脂质纳米颗粒和细胞外囊泡的优劣。最后,本文综述了基因编辑技术在糖生物学研究中的应用,包括糖类物质功能、生物合成与机制,糖蛋白质组学分析,细胞糖芯片的构建和应用以及蛋白质糖基化工程改造。精确基因编辑技术的发展促进了糖类物质的生物合成机制、结构与功能研究,也正在推进转化糖科学研究。

The elucidation of gene functions is a fundamental task in modern biological research,the precisive,efficient,and targeted editing of genes is an indispensable tool for their functional dissection.Over the past 30 years,gene editing has experienced the transformation from homologous recombination repair-based technology to programable nuclease-based method,such as zinc-finger nucleases,transcription activator-like effector nucleases,and CRISPR-associated nucleases.The development of these technologies has greatly advanced the investigation of gene functions and led to the emergence of disruptive technologies for disease treatment.In this manuscript,we first introduced the classification,composition,and working principle of CRISPR-Cas for the immune defense in bacteria.We subsequently focus on the state-of-the-art gene editing tools based on the CRISPR-Cas system including the CRISPRi/CRISPRa,base editors,precursor editors,and the RNA-targeted RCas editing system.We then review the strategies for the delivery of gene editors to the desired target cells/organs,we mainly discuss the pros and cons of adeno-associated viruses,lipid nanoparticles,and extracellular vesicles.Finally,we review the applications of gene editing technologies in glycobiology research,including the function,biosynthesis,and underlying mechanism for the carbohydrates,glycoproteomic analysis,the construction and application of cellular glycan array,and the protein glycoengineering.In conclusion,the development of the precise gene editing technology has significantly promoted the research on the biosynthesis,structure,and function of carbohydrates,which has also advanced the translational aspect of glycoscience research.