Wei Yanlong, Deng Pingping, Mohsin Ali, Yang Yan, Zhou Huayan, Guo Meijin, Fang Hongqing
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Institute of Health Sciences, Anhui University, Economic and Technology Development Zone, Hefei, Anhui, China.
PLoS One. 2017 Oct 24;12(10):e0186891. doi: 10.1371/journal.pone.0186891. eCollection 2017.
The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4-104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3-100%. Additionally, the recombination efficiencies were 2.5-45%, representing an order of magnitude improvement over the electroporation method. For example, for genomic replacement, lycopene expression flux (3.5 kb) was efficiently and precisely integrated into the chromosome, accompanied by replacement of nonessential regions separately into four differently oriented loci. The lycopene production level varied approximately by 5- and 10-fold, corresponding to the integrated position and expression direction, respectively, in the E. coli chromosome.
λ-Red重组系统是一种常用的基因编辑方法。然而，由于DNA片段的电穿孔受限，其应用受到限制。在此，我们提出一种无电穿孔的λ-Red重组方法，其中靶DNA片段在体内由I-CreI内切核酸酶从着陆垫质粒上切除。随后，需要I-SceI内切核酸酶切割染色体和DNA双链断裂修复。通过这种新方法成功实现了无标记缺失和基因组替换。在大肠杆菌DH1基因组中分别删除了8个2.4 - 104.4 kb的非必需区域，选择效率为5.3% - 100%。此外，重组效率为2.5% - 45%，比电穿孔方法提高了一个数量级。例如，对于基因组替换，番茄红素表达通量（3.5 kb）被高效且精确地整合到染色体中，同时非必需区域分别被替换到四个不同方向的位点。在大肠杆菌染色体中，番茄红素的产量水平分别根据整合位置和表达方向变化了约5倍和10倍。
