CRISPRi-Manipulation of Genetic Code Expansion via RF1 for Reassignment of Amber Codon in Bacteria

Bo Zhang,Qing Xia,Jingxian Chen,Demin Zhou,Lihe Zhang,Qi Yang,Huan Xu,Ling Wu,Tianzhuo Yao,Yiming Wu

doi:10.1038/srep20000

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.

Highlights

Despite of the significant achievement on reassignment of UAG as a sense codon, generation of release factor 1 (RF1) knockout strains is usually time-consuming and prevents the application of genetic code expansion
We found that different RF1 repressions were achieved via selection of the targeting sites
The accomplishment of CRISPR interference (CRISPRi)-mediated RF1 repression via a routine bacterial transformation disclosed the fate of the amber codon as either a sense or stop codon, which is dependent on the competing binding of amber codon with either suppressor tRNA or endogenous RF1

Summary

Introduction

Despite of the significant achievement on reassignment of UAG as a sense codon, generation of RF1 knockout strains is usually time-consuming and prevents the application of genetic code expansion. A variety of RF1 repressions were achieved with the incorporation of UAA enhanced but the cell growth lost. Quantitation of RF1 repression versus UAA incorporation and cell growth revealed RF1 as a double-edged sword mediating both beneficial and harmful effects on amber reassignment. The CRISPRi cassette was engineered to be compatible with common vectors for applicable to different proteins and bacteria[16]. The combination of CRISPRi with genetic code expansion via a routine bacterial transformation established a facile and straightforward approach for precise engineering and high production of proteins of interest in bacteria

Methods

Results

Conclusion