CRISPR/Cas12a‐mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus

Abstract

Purple non-sulfur photosynthetic bacteria (PNSB) such as Rhodobacter capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for genome editing and transcriptional regulation in R. capsulatus. Templatefree disruption method mediated by CRISPR/Cas12a reached ~90% editing efficiency when targeting ccoO or nifH gene. When both genes were simultaneously edited, the multiplex editing efficiency reached > 63%. In addition, CRISPR interference (CRISPRi) using deactivated Cas12a was also evaluated using reporter genes egfp and lacZ, and the transcriptional repression efficiency reached ~80%. In summary, our work represents the first report to develop CRISPR/ Cas12a-mediated genome editing and transcriptional regulation in R. capsulatus, which would greatly accelerate PNSB-related researches.

High-Yielding Terpene-Based Biofuel Production in Rhodobacter capsulatus

“…In E. coli , it was reported that zwf knockout led to an increased flux to the glycolytic pathway and tricarboxylic acid (TCA) cycle from glucose . Such a strategy has been adopted in both E. coli and R. sphaeroides , and the lycopene level was improved by 130% and 80%, respectively. , Recently, our group developed a CRSIPR/Cas12a-mediated genome editing tool via a template-free mechanism, and the disruption efficiency reached >90% . When compared to RcBIS6, the disruption of zwf by CRISPR/Cas12a (strain RcBIS7) increased the bisabolene titer up to 181.3 mg/L, which corresponds to a 91% improvement over that of the control (RcBIS6).…”

“…The crRNA of zwf gene was amplified using pY001 (pFnCpf1_full, Addgene plasmid # 69975) as the template with primers LD0-U1-F/zwf-gRNA-R and then cloned into pBRucCas12a (CRISPR/Cas12a tool) through Golden-gate assembly to generate pBRucCas12a-zwf. The crRNAs of phbC , gltB , and gltD were amplified from pY001 with respective primers LD0-U1-F/phbC-gRNA-R, LD0-U2-F/gltB-gRNA-R, and LD0-U3-F/gltD-gRNA-R and then cloned into pBRucCas12a through Golden-gate assembly to generate pBRucCas12a-PGL.…”

“…The interference crRNAs of crtE , ispB , and uppS were amplified using pY001 with primers LD0-U1-F/dcrtE-gRNA-R, LD0-U2-F/dispB-gRNA-R, and LD0-U3-F/duppS-gRNA-R and then cloned into pRKucdCas12a (CRISPR interference, CRISPRi tool) through Golden-gate assembly to give pRKucdCas12a-EBS. In a similar way, the interference crRNAs of crtE , ispB , and crtB were respectively amplified with primers LD0-U1-F/dcrtE-gRNA-R, LD0-U2-F/dispB-gRNA-R, and LD0-U3-F/dcrtB-gRNA-R and cloned into pRKucdCas12a to give pRKucdCas12a-EBB.…”

Analysis of a σ54 Transcription Factor L420P Mutation in Context of Increased Organic Nitrogen Tolerance of Photofermentative Hydrogen Production in Cereibacter sphaeroides Strain 2.4.1 Substrain H2

“…In order to concentrate on the effect of the genotype on the phenotypic observations, we checked which of the proteins from Table 2 are expressed at all at high organic nitrogen concentrations (Figure 4). Only the sigma-54 (σ 54 ) subunit of RNA-polymerase is both expressed under the conditions examined and the SNV exhibits a potential functional effect (Table 2).…”

Potential of Phototrophic Purple Nonsulfur Bacteria to Fix Nitrogen in Rice Fields

“…Template-free disruption method mediated by CRISPR/Cas12a developed for genome editing and transcriptional regulation in R. capsulatus with editing efficiency up to 90% would accelerate genetic construction of PNSB strains [147]. Considering the possibility that alternative nitrogenases in PNSB might efficiently supply hydrogen, a substrate for respiration to reduce oxygen concentration under microaerobic conditions, mutations in vnfA, anfA, or both vnfA and anfA that have the coded proteins adopt their active conformations may be important for efficiently producing hydrogen in the presence of ammonium.…”