Establishing CRISPRi for Programmable Gene Repression and Genome Evolution in Cupriavidus necator

Abstract

Cupriavidus necator H16 is a "Knallgas" bacterium with the ability to utilize various carbon sources and has been employed as a versatile microbial cell factory to produce a wide range of value-added compounds. However, limited genome engineering, especially gene regulation methods, has constrained its full potential as a microbial production platform. The advent of CRISPR/Cas9 technology has shown promise in addressing this limitation. Here, we developed an optimized CRISPR interference (CRISPRi) system for gene repression in C. necator by expressing a codon-optimized deactivated Cas9 (dCas9) and appropriate single guide RNAs (sgRNAs). CRISPRi was proven to be a programmable and controllable tool and could successfully repress both exogenous and endogenous genes. As a case study, we decreased the accumulation of polyhydroxyalkanoate (PHB) via CRISPRi and rewired the carbon fluxes to the synthesis of lycopene. Additionally, by disturbing the expression of DNA mismatch repair gene mutS with CRISPRi, we established CRISPRi-Mutator for genome evolution, rapidly generating mutant strains with enhanced hydrogen peroxide tolerance and robustness in microbial electrosynthesis (MES) system. Our work provides an efficient CRISPRi toolkit for advanced genetic manipulation and optimization of C. necator cell factories for diverse biotechnology applications.

UsingCupriavidus necatorH16 to provide a roadmap for increasing electroporation efficiency in non-model bacteria

“…In addition, this technique can be used for other purposes where high transformation efficiency is required. Some Cas9 plasmids, such as the CRISPRi system recently developed for C. necator H16, are designed for gRNA cloning using BsaI 63 , and a large number of gRNAs could be cloned and tested simultaneously after purification of the natively methylated backbone. However, there are some limitations.…”