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 restriction endonuclease. Recently genome-wide editing creates a new high-performance large fragments cloning methods. For example, CRISPR/cas9 system can identify and cut 20 bp nucleic acid sequences recognition sites used to obtain any desired gene fragments; if combined with Gibson or transformation associated recombination (TAR) assembly technology, these methods can efficiently clone large fragments. This article introduces large fragments cloning technology by classification, then proposes the choice criteria of methods for cloning gene fragments of different sizes.
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。.
Keywords: CRISPR/cas9; Gibson assembly; cloning large gene cluster; synthetic biology.