Fast genome editing in <i>Bacillus subtilis</i>

Wu, Guo; Drufva, Erin E.; Wu, Kang

doi:10.1002/elsc.201800164

Cited by 7 publications

(5 citation statements)

References 28 publications

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“…However, whenever high‐efficiency transformation and other factors permit, vector‐free KO approach should be considered as an alternative offering lower off‐target mutagenesis probability. Vector‐free AECs in previous studies were Fusion PCR products circularized by ligation (Gomaa et al, 2017 ) and linear GA products (Wu et al, 2019 ). Here we combined these two approaches: we used GA as a more convenient alternative to Fusion PCR technique (Huang & Wilks, 2017 ; Rudenko & Barnes, 2018 ), but employed it to generate circular constructs/products, as they are more stable compared to linear DNA.…”

Section: Resultsmentioning

confidence: 99%

Transforming the untransformable with knockout minicircles: High‐efficiency transformation and vector‐free allelic exchange knockout in the fish pathogen Photobacterium damselae

Rudenko,

Baseggio,

McGuigan

et al. 2023

MicrobiologyOpen

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Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange via homologous recombination is a generic method of targeted gene editing in bacteria. However, generally applicable protocols are lacking, and suboptimal approaches are often used for nonstandard but epidemiologically important species. Photobacterium damselae subsp. piscicida (Pdp) is a primary pathogen of fish in aquaculture and has been considered hard to transform since the mid‐1990s. Consequently, conjugative transfer of RK2/RP4 suicide vectors from Escherichia coli S17‐1/SM10 donor strains, a system prone to off‐target mutagenesis, was used to deliver the allelic exchange DNA in previous studies. Here we have achieved efficient electrotransformation in Pdp using a salt‐free highly concentrated sucrose solution, which performs as a hypertonic wash buffer, cryoprotectant, and electroporation buffer. High‐efficiency transformation has enabled vector‐free mutagenesis for which we have employed circular minimalistic constructs (knockout minicircles) containing only allelic exchange essentials that were generated by Gibson assembly. Preparation of competent cells using sucrose and electroporation/integration of minicircles had virtually no detectable off‐target promutagenic effect. In contrast, a downstream sacB selection apparently induced several large deletions via mobilization of transposable elements. Electroporation of minicircles into sucrose‐treated cells is a versatile broadly applicable approach that may facilitate allelic exchange in a wide range of microbial species. The method permitted inactivation of a primary virulence factor unique to Pdp, apoptogenic toxin AIP56, demonstrating the efficacy of minicircles for difficult KO targets located on the high copy number of small plasmids.

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Section: Resultsmentioning

confidence: 99%

Transforming the untransformable with knockout minicircles: High‐efficiency transformation and vector‐free allelic exchange knockout in the fish pathogen Photobacterium damselae

Rudenko,

Baseggio,

McGuigan

et al. 2023

MicrobiologyOpen

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“…Various high efficiency, simultaneous multilocus integration methods based on two-plasmid CRISPR-Cas9 systems have been implemented in E. coli [42,43], yeasts [44,45], and fungi [46,47], making them suitable host cells to be used as a chassis in the field of synthetic biology. However, although the emergence of the CRISPR/ Cas9 system has led to numerous new applications and developments in B. subtilis [29,30,[48][49][50][51], efficient methods for simultaneous gene insertion in B. subtilis…”

Section: Discussionmentioning

confidence: 99%

Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system

Ferrando

Filluelo

Zeigler³

et al. 2023

Microb Cell Fact

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Background Despite recent advances in genetic engineering tools for effectively regulating and manipulating genes, efficient simultaneous multigene insertion methods have not been established in Bacillus subtilis. To date, multilocus integration systems in B. subtilis, which is one of the main industrial enzyme producers and a GRAS (generally regarded as safe) microbial host, rely on iterative rounds of plasmid construction for sequential insertions of genes into the B. subtilis chromosome, which is tedious and time consuming. Results In this study, we present development and proof-of-concept of a novel CRISPR-Cas9-based genome-editing strategy for the colorimetric detection of one-step multiple gene insertion in B. subtilis. First, up to three copies of the crtMN operon from Staphylococcus aureus, encoding a yellow pigment, were incorporated at three ectopic sites within the B. subtilis chromosome, rendering engineered strains able to form yellow colonies. Second, a single CRISPR-Cas9-based plasmid carrying a highly specific single guide RNA (sgRNA) targeting crtMN operon and a changeable editing template was constructed to facilitate simultaneous insertion of multiple gene-copies through homology-directed repair (HDR). Upon transformation of engineered strains with engineered plasmids, strains harboring up to three gene copies integrated into the chromosome formed white colonies because of the removal of the crtMN operon, clearly distinguishable from yellow colonies harboring undesired genetic modifications. As a result, construction of a plasmid-less, marker-free, high-expression stable producer B. subtilis strain can be completed in only seven days, demonstrating the potential that the implementation of this technology may bring for biotechnology purposes. Conclusions The novel technology expands the genome-editing toolset for B. subtilis and means a substantial improvement over current methodology, offering new application possibilities that we envision should significantly boost the development of B. subtilis as a chassis in the field of synthetic biology.

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“…All plasmids, except p7INT ( Supplementary Figure S1B ) and p7INT.1, were linearized using Alw 44I prior to transformation into S. pyogenes SF370 to favor double-crossover recombination ( Heap et al, 2012 ; Wu et al, 2019 ). Integration of the plasmids was verified by PCR and the integration sites and distinct virulence genes ( ropB , csrRS , mga ) were sequenced using Sanger sequencing.…”

Section: Methodsmentioning

confidence: 99%

Expanding the genetic toolbox for the obligate human pathogen Streptococcus pyogenes

Lautenschläger,

Schmidt,

Schiffer

et al. 2024

Front. Bioeng. Biotechnol.

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Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P23 showing the strongest activity and the synthetic promoter PxylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.

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Fast genome editing in Bacillus subtilis

Cited by 7 publications

References 28 publications

Transforming the untransformable with knockout minicircles: High‐efficiency transformation and vector‐free allelic exchange knockout in the fish pathogen Photobacterium damselae

Transforming the untransformable with knockout minicircles: High‐efficiency transformation and vector‐free allelic exchange knockout in the fish pathogen Photobacterium damselae

Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system

Expanding the genetic toolbox for the obligate human pathogen Streptococcus pyogenes

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