CRISPR/Cas9–Mediated Genome Editing for Pseudomonas fulva, a Novel Pseudomonas Species with Clinical, Animal, and Plant–Associated Isolates

Abstract

As one of the most widespread groups of Gram–negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant–associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time–consuming and laborious endeavor. Here, by using the CRISPR–Cas system, a versatile gene–editing tool with a two–plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic–related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked–in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species.

Genome editing in the edible fungus Poria cocos using CRISPR-Cas9 system integrating genome-wide off-target prediction and detection

“…The off-target effects are commonly due to PAM and sgRNA mismatches. Currently, various bioinformatics tools have been developed to help predict and reduce off-target modifications ( Zhang et al, 2022 ). In this study, the potential off-target sites of CRISPR/Cas9 were predicted through 12 nucleotide sequences of sgRNA and PAM sites by aligning the sgRNA sequences with reference genome sequences using Tbtools software to search mismatch.…”

Rice-fish symbiosis improves the muscle nutrition and intestinal flora diversity of tilapia

“…At the same time, Ralstonia is an important inflammatory factor in the intestine. [27][28][29][30][31][32] The study further showed that the abundance of Pseudomonas and Ralstonia in the R group was significantly higher than in the T group (p<0.05). We speculate that the composition of tilapia intestinal flora in the rice-fish symbiosis mode is more conducive to fish health.…”

Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species

“…Although Pseudomonas strains have an established role in the biotechnology field ( 26–28 ), their potential could be enhanced by further exploiting their unique traits and improving our ability to modify them using genetically engineering ( 29–31 ). Although improving intrinsic lifestyle characteristics such as tolerance, handling, growth and substrate consumption rates remains challenging, the incredible progress of genetic engineering during the last decades sets the stage for efficient tailoring of microbial metabolism for a large array of bioconversions, including, for example, the assimilation of non-canonical nutrients and C1 compounds.…”