Fig 1.
Overview of steps involved in the cross-species CRISPR-Cas9 genome editing strategy (not drawn to scale).
(A): Chromosomal integration of the landing pad vector by mariner-based transposon system. (B): Chromosome integration of the Cas9 and RecET via RMCE. (C): Chromosome integration of the sgRNA and donor template via RMCE, followed by genome editing at the specific target site. The orientation of lox sites was kept consistent in all the vectors. IR is the transposon-specific inverted repeats. oriT is the origin of transfer. KanR and AprR are antibiotic resistance cassettes for kanamycin and apramycin, respectively. OriS, oriV and R6K are different replication origins.
Table 1.
Summary of successful genome editing in four different bacteria species.
Fig 2.
Construction of Cas9-mediated S. oneidensis flaG gene deletion mutants.
(A): The editing schematic diagram and screening primers are shown for deletion of the flaG gene. (B): The phenotype of different S. oneidensis strains. WT, wild type. Cas9, the wild type strain harboring Cre, Cas9, RecET and AprR in the chromosome. Cas9+sgRNA, the wild type strain harboring donor template, sgRNA, KanR, Cas9, RecET and AprR in the chromosome. (C): PCR screening of the initial transconjugant colonies. L: DNA ladder. WT: wild type as positive control. 1–16: 16 randomly picked colonies.
Fig 3.
The Cas9-assisted deletion editing efficiency in P. luminescens subsp. laumondii TT01.
Edited: colony PCR yielding an amplicon of edited cells. Chimeric: colony PCR yielding mixed amplicons with both edited and unedited cells. Unedited: PCR only yielding an amplicon of unedited cells.
Fig 4.
Removing the editing machinery in P. luminescens.
(A): The curing schematic diagram and screening primers are shown for curing the editing machinery (not drawn to scale). (B): The structure of the curing plasmid. (C): PCR screening after curing (the larger band in isolate 6 is a non-specific amplification). L: DNA ladder. WT: wild type as positive control. 1–16: 16 randomly picked colonies. (D) The sequence alignment analysis of three strains at two specific loci on the chromosome. CGGAZ, CGGBB, and CGGBG denote as reads generated from a wild-type strain, an edited strain carrying the landing pad, Cas9, and sgRNA, and an edited strain with landing pad removed, respectively. The red vertical lines indicate the 3 lox sites in the landing pad.