摘要
基因组定点编辑(site-specific genome editing)是指在基因组水平上对生物DNA序列进行定点改造的遗传操作技术,其在基因功能解析、动植物遗传改良和新品种培育等方面具有重大的应用价值。基因组定点编辑工作原理是利用序列特异性核酸内切酶(sequence-specific nucleases,SSNs)在基因组靶定位置切割DNA双链,造成DNA双链断裂(DNA double-strand breaks,DSBs),并通过非同源末端连接(non-homologous end joining,NHEJ)或同源重组(homology-directed repair,HDR)的DNA修复途径在基因组特定位点造成靶标基因的碱基插入、缺失或DNA片段替换,从而实现基因组的定点改造。目前,已成功应用于作物遗传改良的SSNs主要包括锌指核酸酶(Zinc finger nucleases,ZFNs)、类转录激活因子效应物核酸酶(transcription activator-like effector nucleases,TALENs)、成簇的规律间隔的短回文重复序列及其相关系统(clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins,CRISPR/Cas system)。从发展态势看,基于CRISPR/Cas系统的基因组编辑技术必将成为作物遗传改良和分子设计育种的核心技术之一。论文简要概述了ZFNs、TALENs和CRISPR/Cas系统这3种基因组编辑的技术背景及工作原理;结合案例重点综述上述3种技术在作物产量、品质、抗病性、抗逆性改良及水稻雄性不育系创制上的研究进展;详细梳理基于CRISPR/Cas的植物基因组单碱基编辑系统和DNA-free植物基因组编辑系统的技术创新和应用;比较分析3种技术的优缺点,并提出基因组编辑技术应用于作物遗传改良的一般原则;介绍了美国和欧盟等对基因编辑技术及其产品安全监管和商业化应用的政策法规,及业界人士对基因编辑作物提出的监管框架协议;最后,针对基因编辑技术自身的技术优势和缺陷,讨论该技术应用于作物遗传改良和分子育种的机遇和挑战。
Site-specific genome editing refers to the genetic manipulation techniques that can precisely modify DNA sequences at the genomic level, which holds a great application value in analysis of gene function, plant genetic improvement or development of new varieties. The general working principle of genome editing is that the sequence-specific nucleases(SSNs) are used to create DNA double-strand breaks(DSBs) at the genomic target sites that will be repaired through non-homologous end joining(NHEJ) and/or homology-directed repair(HDR) pathways, resulting in nucleotide insertion, deletion, or DNA fragment replacement in the targeted gene(s). Nowadays, genome editing technologies including Zinc finger nucleases(ZFNs), transcription activator-like effector nucleases(TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins(CRISPR/Cas)system have been successfully applied in genetic improvement of crops. From the perspective of development trend, CRISPR/Cas system-based genome editing technology will become the core technology in genetic improvement and molecular design breeding of crops. This review starts with brief introduction of the concepts and working principles of ZFNs, TALENs and CRISPR/Cas system, and focus on the recent progress of genome editing-based crop improvement of yield, quality, disease resistance, abiotic stress resistance and rice male sterile line development. Then the authors sorted out the technological innovation and application of CRISPR/Cas-based single-base editing for plant genomes and the DNA-free plant genome editing system in details. After comparing the advantages and disadvantages of different genome editing techniques, some general principles of adoption of genome editing technologies in crop improvement were put forward. Finally, the regulations on genome-edited crops, as well as the opportunities and challenges of genome editing-based crop breeding were discussed.
引文
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