Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum.

Jizhong Zhou,Zhili He,Yongchao Li,Tao Xu,Joy D. Van Nostrand

doi:10.3389/fmicb.2017.01744

Abstract

Essential gene functions remain largely underexplored in bacteria. Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing; however, its genetic manipulation to reduce the formation of less-valuable acetate is technically challenging due to the essentiality of acetate-producing genes. Here we developed a Cas9 nickase-assisted chromosome-based RNA repression to stably manipulate essential genes in C. cellulolyticum. Our plasmid-based expression of antisense RNA (asRNA) molecules targeting the phosphotransacetylase (pta) gene successfully reduced the enzymatic activity by 35% in cellobiose-grown cells, metabolically decreased the acetate titer by 15 and 52% in wildtype transformants on cellulose and xylan, respectively. To control both acetate and lactate simultaneously, we transformed the repression plasmid into lactate production-deficient mutant and found the plasmid delivery reduced acetate titer by more than 33%, concomitant with negligible lactate formation. The strains with pta gene repression generally diverted more carbon into ethanol. However, further testing on chromosomal integrants that were created by double-crossover recombination exhibited only very weak repression because DNA integration dramatically lessened gene dosage. With the design of a tandem repetitive promoter-driven asRNA module and the use of a new Cas9 nickase genome editing tool, a chromosomal integrant (LM3P) was generated in a single step and successfully enhanced RNA repression, with a 27% decrease in acetate titer on cellulose in antibiotic-free medium. These results indicate the effectiveness of tandem promoter-driven RNA repression modules in promoting gene repression in chromosomal integrants. Our combinatorial method using a Cas9 nickase genome editing tool to integrate the gene repression module demonstrates easy-to-use and high-efficiency advantages, paving the way for stably manipulating genes, even essential ones, for functional characterization and microbial engineering.

Highlights

Essential genes are indispensable for building up the chassis of living organisms (Glass et al, 2006), and accounts for 5–80% of bacterial genomes (Gao and Zhang, 2011)
Considering the easy-to-use and highly efficient advantages of CRISPR/Cas9based genome editing tools (Xu et al, 2014, 2015) and the simplicity and universality of antisense RNA-mediated repression (Thomason and Storz, 2010), here we propose a combination of these two methods using Cas9 technology to integrate antisense RNA modules into the genome
Its 5 transcriptional region with a length of approximately 120 bp was inserted in a reverse orientation under the control of a ferredoxin promoter to produce antisense RNA (asRNA) which will interfere with the stability and translation of target transcripts (Figure 1B) (Thomason and Storz, 2010)

Summary

Introduction

Essential genes are indispensable for building up the chassis of living organisms (Glass et al, 2006), and accounts for 5–80% of bacterial genomes (Gao and Zhang, 2011). There are three major approaches available for targeted gene repression in bacteria, including antisense RNA (asRNA)-mediated repression (Desai and Papoutsakis, 1999; Perret et al, 2004; Thomason and Storz, 2010), Hfp-dependent RNA repression (Man et al, 2011; Na et al, 2013) and nuclease-null Cas9-mediated repression (which is named CRISPRi) (Bikard et al, 2013; Qi et al, 2013). Knock-down mutants can be created in a single step with features that are plasmid-independent and can be sustained without using antibiotics

Methods

Results

Conclusion