Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China.
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, 100071, China.
Biotechnol Lett. 2020 Aug;42(8):1457-1465. doi: 10.1007/s10529-020-02856-7. Epub 2020 Mar 4.
To develop a convenient double-locus scarless genome editing system in Escherichia coli, based on the type II Streptococcus pyogenes CRISPR/Cas9 and λ Red recombination cassette.
A two-plasmid genome editing system was constructed. The large-sized plasmid harbors the cas9 and λ Red recombination genes (gam, bet, and exo), while the small-molecular plasmid can simultaneously express two different gRNAs (targeting genome RNAs). The recombination efficiency was tested by targeting the galK, lacZ, and dbpA genes in E. coli with ssDNA or dsDNA. Resulting concurrent double-locus recombination efficiencies were 88 ± 5.5% (point mutation), 39.7 ± 4.3% (deletion/insertion), and 57.8 ± 3.4%-58.5 ± 4.1% (mixed point and deletion/insertion mutation), depending on 30 (ssDNA) or 40 bp (dsDNA) homologous side arms employed. In addition, the curing efficiency of the guide plasmid expressing gRNAs for negative selection was higher (96 ± 3% in 4 h) than the help plasmid carrying cas9 and λ Red (92 ± 2% in 9 h).
The new editing system is convenient and efficient for simultaneous double-locus recombination in the genome and should be favorable for high-throughput multiplex genome editing in synthetic biology and metabolic engineering.
基于 II 型酿脓链球菌 CRISPR/Cas9 和 λ Red 重组盒，开发一种简便的大肠杆菌无疤痕双基因座基因组编辑系统。
构建了一个双质粒基因组编辑系统。大质粒携带 cas9 和 λ Red 重组基因（gam、bet 和 exo），而小质粒可同时表达两种不同的 gRNA（靶向基因组 RNA）。通过 ssDNA 或 dsDNA 靶向大肠杆菌的 galK、lacZ 和 dbpA 基因测试重组效率。结果，点突变的并发双基因座重组效率为 88±5.5%，缺失/插入的为 39.7±4.3%，混合点突变和缺失/插入突变的为 57.8±3.4%-58.5±4.1%，具体取决于 30（ssDNA）或 40 bp（dsDNA）同源臂的使用。此外，用于负选择的表达 gRNA 的指导质粒的消除效率更高（4 h 时为 96±3%），而携带 cas9 和 λ Red 的辅助质粒的消除效率较低（9 h 时为 92±2%）。
新的编辑系统在基因组中进行同时双基因座重组既方便又高效，应该有利于合成生物学和代谢工程中的高通量多重基因组编辑。
