文章基本信息

标题：CRISPRi-Manipulation of Genetic Code Expansion via RF1 for Reassignment of Amber Codon in Bacteria
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作者：Bo Zhang ; Qi Yang ; Jingxian Chen 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2016
卷号：6
DOI：10.1038/srep20000
出版社：Springer Nature
摘要：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.