title: Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4.
abstract: The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.
authors.affiliation: a Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences , Sichuan University , Chengdu , Sichuan , China.|b Sichuan Key Laboratory of Molecular Biology and Biotechnology , Sichuan University , Chengdu , Sichuan , China.
authors.forename: Qing-Hua
authors.initials: QH
authors.lastname: Cao
authors.affiliation: a Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences , Sichuan University , Chengdu , Sichuan , China.|b Sichuan Key Laboratory of Molecular Biology and Biotechnology , Sichuan University , Chengdu , Sichuan , China.
authors.forename: Huan-Huan
authors.initials: HH
authors.lastname: Shao
authors.affiliation: a Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences , Sichuan University , Chengdu , Sichuan , China.|b Sichuan Key Laboratory of Molecular Biology and Biotechnology , Sichuan University , Chengdu , Sichuan , China.
authors.forename: Hui
authors.initials: H
authors.lastname: Qiu
authors.affiliation: a Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences , Sichuan University , Chengdu , Sichuan , China.|b Sichuan Key Laboratory of Molecular Biology and Biotechnology , Sichuan University , Chengdu , Sichuan , China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: a Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences , Sichuan University , Chengdu , Sichuan , China.|b Sichuan Key Laboratory of Molecular Biology and Biotechnology , Sichuan University , Chengdu , Sichuan , China.
authors.forename: Yi-Zheng
authors.initials: YZ
authors.lastname: Zhang
authors.affiliation: a Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Sciences , Sichuan University , Chengdu , Sichuan , China.|b Sichuan Key Laboratory of Molecular Biology and Biotechnology , Sichuan University , Chengdu , Sichuan , China.
authors.forename: Xue-Mei
authors.initials: XM
authors.lastname: Tan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D055786:Gene Knockout Techniques / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D017355:Zymomonas / Q000235:genetics* / Q000254:growth & development
chemical_list: D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR/Cas9 system; Zymomonas mobilis; expression plasmid; plasmid curing