A double-locus scarless genome editing system in Escherichia coli.

Autor: Liu H; Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China., Hou G; Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China., Wang P; Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, 100071, China., Guo G; Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China., Wang Y; Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China., Yang N; Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China., Rehman MNU; Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China., Li C; Department of Medical Genetics and Developmental Biology, School of Basic Medical Science, Capital Medical University, Beijing, 100069, China., Li Q; Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China., Zheng J; Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China. jiping.zheng@hainu.edu.cn., Zeng J; Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life Sciences and Pharmacy, Hainan University, Haikou, 570228, China. jifengzeng@126.com., Li S; Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, 100071, China. lishanhu6@163.com.
Jazyk: angličtina
Zdroj: Biotechnology letters [Biotechnol Lett] 2020 Aug; Vol. 42 (8), pp. 1457-1465. Date of Electronic Publication: 2020 Mar 04.
DOI: 10.1007/s10529-020-02856-7
Abstrakt: Objective: 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.
Results: 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).
Conclusions: 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.
Databáze: MEDLINE
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