title: CRISPRi-Manipulation of Genetic Code Expansion via RF1 for Reassignment of Amber Codon in Bacteria.
abstract: The precise engineering of proteins in bacteria via the amber codon has been hampered by the poor incorporation of unnatural amino acid (UAA). Here we explored the amber assignment as a sense codon for UAA by CRISPRi targeting release factor 1 (RF1). Scanning of RF1 gene with sgRNAs identified target loci that differentiate RF1 repressions. Quantitation of RF1 repressions versus UAA incorporation indicated an increasing interrelation with the amber reassignment maximized upon RF1 knockdown to ~30%, disclosing the beneficial role of RF1 in amber assignment. However, further RF1 repression reversed this trend resulting from the detrimental effects on host cell growth, disclosing the harmful aspect of RF1 in reassignment of the amber codon. Our data indicate RF1 as a switch manipulating genetic code expansion and pave a direction via CRISPRi for precise engineering and efficient production of proteins in bacteria.
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Chemical Biology, Peking University, Beijing 100191, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences.
authors.forename: Jingxian
authors.initials: J
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences.
authors.forename: Ling
authors.initials: L
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences.
authors.forename: Tianzhuo
authors.initials: T
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences.
authors.forename: Huan
authors.initials: H
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences.
authors.forename: Lihe
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Chemical Biology, Peking University, Beijing 100191, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xia
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences.
authors.forename: Demin
authors.initials: D
authors.lastname: Zhou
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018388:Codon, Terminator; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D005815:Genetic Code; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D018388:Codon, Terminator; D010454:Peptide Termination Factors
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