基于基因组编辑技术的大片段克隆新策略.PDFVIP

生 物 工 程 学 报 曾哲 等/基于基因组编辑技术的大片段克隆新策略 401 Chinese Journal of Biotechnology /cjbcn April 25, 2016, 32(4): 401−408 DOI: 10.13345/j.cjb.150491 ©2016 Chin J Biotech, All rights reserved 综 述 曾哲，任晓丹，杨晟 200032 , , . . , 2016, 32(4): 401–408. Zeng Z, Ren XD, Yang S. New methods for cloning large gene clusters based on CRISPR/cas9. Chin J Biotech, 2016, 32(4): 401–408. 摘 要: 基因组大片段克隆技术是合成生物学研究领域关键的使能技术。传统的大片段克隆技术获取目的大 片段的手段存在各种缺陷，比如随机建库克隆需要依靠高通量筛选；PCR 难以扩增 10 kb 以上片段，从小片段 拼装费时费力且突变率高；基于限制性酶切连接难以找到片段两端适宜的限制性内切酶酶切位点。最近全基因 组合成等前沿研究创造了全新的高性能大片段克隆方法，比如 CRISPR/cas9 系统中cas9 可识别并切割 20 bp 核酸序列解决了识别位点设计难题，可用来获取任意目的基因片段；组合 Gibson 或者酵母偶联重组技术组装 技术，可高效克隆大片段基因。本文将分类介绍基因组大片段克隆技术，并提出适用不同尺度大小基因克隆的 技术选择参考标准。 : 大片段克隆，合成生物学，基因组编辑，Gibson 组装 New methods for cloning large gene clusters based on CRISPR/cas9 Zhe Zeng, Xiaodan Ren, and Sheng Yang CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (CAS), Shanghai 200032, China Abstract: Cloning of large genomic sequences is an enabling technology in synthetic biology. To obtain large gene fragments, traditional cloning methods are faced with various defects, for instance, random library cloning relies always on high-throughput screening. It is difficult to get gene fragments more than 10 kb by PCR amplification. Assembly of small fragments is labor intensive with high mutation rates. It is difficult to find suitable cleavage sites on the fragment ends by Received: November 19, 2015; Accepted: January 22, 2016 Supported by: National Natural Science Foundation of China