CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa

10.1101/2021.08.06.455192 ◽

2021 ◽

Author(s):

Meliawati Meliawati ◽

Christa Teckentrup ◽

Jochen Schmid

Keyword(s):

Genetic Engineering ◽

Genome Editing ◽

Related Species ◽

Paenibacillus Polymyxa ◽

Genomic Region ◽

Large Cluster ◽

Future Research ◽

Engineering Research ◽

Crispr System ◽

Gene Knockouts

Clustered regularly interspaced short palindromic repeats (CRISPR) system has rapidly advanced genetic engineering research. The system has been applied for different genetic engineering purposes in multiple organisms including the quite rarely explored Paenibacillus polymyxa. Only limited studies on CRISPR-based system have been described for this highly interesting and versatile bacterium. Here, we demonstrated the utilization of a Cas9-based system to realize 32.8 kb deletion of genomic region by using a single targeting sgRNA. Large cluster deletion was successfully performed with remarkable efficiency of 97 %. Furthermore, we also exploited the system for multiplexing by editing of two distantly located genes at once. We investigated double gene knockouts as well as simultaneous gene integrations and reached editing efficiencies of 78 % and 50 %, respectively. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.

Download Full-text

Cytosine Base Editor-Mediated Multiplex Genome Editing to Accelerate Discovery of Novel Antibiotics in Bacillus subtilis and Paenibacillus polymyxa

Frontiers in Microbiology ◽

10.3389/fmicb.2021.691839 ◽

2021 ◽

Vol 12 ◽

Author(s):

Man Su Kim ◽

Ha-Rim Kim ◽

Da-Eun Jeong ◽

Soo-Keun Choi

Keyword(s):

Bacillus Subtilis ◽

Genome Editing ◽

Paenibacillus Polymyxa ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Antimicrobial Activities ◽

Biosynthetic Gene ◽

Single Nucleotide Variants ◽

New Antibiotics ◽

Bacteria And Fungi

Genome-based identification of new antibiotics is emerging as an alternative to traditional methods. However, uncovering hidden antibiotics under the background of known antibiotics remains a challenge. To over this problem using a quick and effective genetic approach, we developed a multiplex genome editing system using a cytosine base editor (CBE). The CBE system achieved simultaneous double, triple, quadruple, and quintuple gene editing with efficiencies of 100, 100, 83, and 75%, respectively, as well as the 100% editing efficiency of single targets in Bacillus subtilis. Whole-genome sequencing of the edited strains showed that they had an average of 8.5 off-target single-nucleotide variants at gRNA-independent positions. The CBE system was used to simultaneously knockout five known antibiotic biosynthetic gene clusters to leave only an uncharacterized polyketide biosynthetic gene cluster in Paenibacillus polymyxa E681. The polyketide showed antimicrobial activities against gram-positive bacteria, but not gram-negative bacteria and fungi. Therefore, our findings suggested that the CBE system might serve as a powerful tool for multiplex genome editing and greatly accelerating the unraveling of hidden antibiotics in Bacillus and Paenibacillus species.

Download Full-text

Tailor-made exopolysaccharides—CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa

Synthetic Biology ◽

10.1093/synbio/ysx007 ◽

2017 ◽

Vol 2 (1) ◽

Cited By ~ 19

Author(s):

Marius Rütering ◽

Brady F Cress ◽

Martin Schilling ◽

Broder Rühmann ◽

Mattheos A G Koffas ◽

...

Keyword(s):

Genome Editing ◽

Gene Deletion ◽

State Of The Art ◽

Paenibacillus Polymyxa ◽

Vector System ◽

Wild Type ◽

Monomer Composition ◽

Homology Directed Repair ◽

Highly Efficient ◽

Micro Organisms

Abstract Application of state-of-the-art genome editing tools like CRISPR-Cas9 drastically increase the number of undomesticated micro-organisms amenable to highly efficient and rapid genetic engineering. Adaptation of these tools to new bacterial families can open up entirely new possibilities for these organisms to accelerate as biotechnologically relevant microbial factories, also making new products economically competitive. Here, we report the implementation of a CRISPR-Cas9 based vector system in Paenibacillus polymyxa, enabling fast and reliable genome editing in this host. Homology directed repair allows for highly efficient deletions of single genes and large regions as well as insertions. We used the system to investigate the yet undescribed biosynthesis machinery for exopolysaccharide (EPS) production in P. polymyxa DSM 365, enabling assignment of putative roles to several genes involved in EPS biosynthesis. Using this simple gene deletion strategy, we generated EPS variants that differ from the wild-type polymer not only in terms of monomer composition, but also in terms of their rheological behavior. The developed CRISPR-Cas9 mediated engineering approach will significantly contribute to the understanding and utilization of socially and economically relevant Paenibacillus species and extend the polymer portfolio.

Download Full-text