Fragment Exchange Plasmid Tools for CRISPR/Cas9-Mediated Gene Integration and Protease Production in Bacillus subtilis

ABSTRACT Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool for targeted genome editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial species. A collection of vectors compatible with high-throughput (HTP) fragment exchange (FX) cloning for heterologous expression in Escherichia coli and Bacillus was previously developed. This vector catalogue was through this work supplemented with editing plasmids for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to the stable integration of the gene into the amyE locus of a seven-protease-deficient KO7 strain. The newly engineered B. subtilis strain allowed the successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering, and contribute to a cloning platform for a quick transition from HTP enzyme expression to production through the fermentation of industrially relevant B. subtilis and related strains. IMPORTANCE We complemented a cloning platform with new editing plasmids that allow a quick transition from high-throughput cloning and the expression of new enzymes to the stable integration of genes for the production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome-editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus’ capacity to sporulate and by introducing the stable expression of subtilisin. The tools should be useful to engineer B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry-relevant host.

Download Full-text

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Applied and Environmental Microbiology ◽

10.1128/aem.01453-16 ◽

2016 ◽

Vol 82 (17) ◽

pp. 5421-5427 ◽

Cited By ~ 102

Author(s):

Josef Altenbuchner

Keyword(s):

Bacillus Subtilis ◽

Genome Editing ◽

Genome Engineering ◽

Shuttle Vector ◽

Temperature Sensitive ◽

Target Sequence ◽

Target Specificity ◽

Content Type ◽

A Genome ◽

Single Plasmid

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system fromStreptococcus pyogeneshas recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing ofBacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication forEscherichia coli, the temperature-sensitive replication origin of plasmid pE194tsforB. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries thecas9gene under the control of theB. subtilismannose-inducible promoter PmanPand a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5′ end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in theB. subtilischromosome and by repair of thetrpC2mutation ofB. subtilis168.IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing ofB. subtilisovercomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

Download Full-text

Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides

mSphere ◽

10.1128/msphere.00099-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 17

Author(s):

Peter B. Otoupal ◽

Masakazu Ito ◽

Adam P. Arkin ◽

Jon K. Magnuson ◽

John M. Gladden ◽

...

Keyword(s):

Genome Editing ◽

Gene Disruption ◽

Genome Engineering ◽

Carbon Sources ◽

Rhodosporidium Toruloides ◽

Optimized Design ◽

Content Type ◽

Wide Range ◽

Fuels And Chemicals ◽

Microbial Biofuel

ABSTRACT Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides’ engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides. Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism. IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast’s production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

Download Full-text

A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis

Microbiology Spectrum ◽

10.1128/spectrum.00754-21 ◽

2021 ◽

Author(s):

Ankita J. Sachla ◽

Alexander J. Alfonso ◽

John D. Helmann

Keyword(s):

Bacillus Subtilis ◽

Genome Editing ◽

Model Organism ◽

Gram Positive ◽

Content Type ◽

Guide Rna ◽

Simplified Method ◽

Repair Template

Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis , they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application.

Download Full-text

Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System

Applied and Environmental Microbiology ◽

10.1128/aem.00233-17 ◽

2017 ◽

Vol 83 (10) ◽

Cited By ~ 47

Author(s):

Shaohua Wang ◽

Sheng Dong ◽

Pixiang Wang ◽

Yong Tao ◽

Yi Wang

Keyword(s):

Genome Editing ◽

Mutation Rate ◽

Genome Engineering ◽

Engineering System ◽

Production Mechanism ◽

Butanol Production ◽

Content Type ◽

Solvent Production ◽

Double Deletion ◽

Butyrate Production

ABSTRACT Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk, two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P J23119 , we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii. Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum. Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the pta buk double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations. IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production.

Download Full-text

CRISPR-Cas9D10A Nickase-Assisted Genome Editing in Lactobacillus casei

Applied and Environmental Microbiology ◽

10.1128/aem.01259-17 ◽

2017 ◽

Vol 83 (22) ◽

Cited By ~ 54

Author(s):

Xin Song ◽

He Huang ◽

Zhiqiang Xiong ◽

Lianzhong Ai ◽

Sheng Yang

Keyword(s):

Genome Editing ◽

Lactobacillus Casei ◽

Genome Engineering ◽

Fluorescent Protein ◽

Genetic Manipulation ◽

Gene Knockout ◽

Single Gene ◽

Egfp Expression ◽

Broad Host Range ◽

Content Type

ABSTRACT Lactobacillus casei has drawn increasing attention as a health-promoting probiotic, while effective genetic manipulation tools are often not available, e.g., the single-gene knockout in L. casei still depends on the classic homologous recombination-dependent double-crossover strategy, which is quite labor-intensive and time-consuming. In the present study, a rapid and precise genome editing plasmid, pLCNICK, was established for L. casei genome engineering based on CRISPR-Cas9D10A. In addition to the P23-Cas9D10A and Pldh-sgRNA (single guide RNA) expression cassettes, pLCNICK includes the homologous arms of the target gene as repair templates. The ability and efficiency of chromosomal engineering using pLCNICK were evaluated by in-frame deletions of four independent genes and chromosomal insertion of an enhanced green fluorescent protein (eGFP) expression cassette at the LC2W_1628 locus. The efficiencies associated with in-frame deletions and chromosomal insertion is 25 to 62%. pLCNICK has been proved to be an effective, rapid, and precise tool for genome editing in L. casei, and its potential application in other lactic acid bacteria (LAB) is also discussed in this study. IMPORTANCE The lack of efficient genetic tools has limited the investigation and biotechnological application of many LAB. The CRISPR-Cas9D10A nickase-based genome editing in Lactobacillus casei, an important food industrial microorganism, was demonstrated in this study. This genetic tool allows efficient single-gene deletion and insertion to be accomplished by one-step transformation, and the cycle time is reduced to 9 days. It facilitates a rapid and precise chromosomal manipulation in L. casei and overcomes some limitations of previous methods. This editing system can serve as a basic technological platform and offers the possibility to start a comprehensive investigation on L. casei. As a broad-host-range plasmid, pLCNICK has the potential to be adapted to other Lactobacillus species for genome editing.

Download Full-text

Faculty Opinions recommendation of High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726346764.793519161 ◽

2016 ◽

Author(s):

Vijay Tiwari ◽

Amitava Basu

Keyword(s):

High Resolution ◽

Genome Editing ◽

High Throughput ◽

Protein Localization ◽

Mammalian Brain ◽

High Resolution Mapping ◽

Resolution Mapping

Download Full-text

Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

aBIOTECH ◽

10.1007/s42994-021-00042-5 ◽

2021 ◽

Author(s):

Jun Li ◽

Yan Li ◽

Ligeng Ma

Keyword(s):

Functional Genomics ◽

Genome Editing ◽

Functional Annotation ◽

Genome Engineering ◽

Agronomic Traits ◽

Wheat Breeding ◽

Breeding Programs ◽

Base Editing ◽

The World ◽

World Food Security

AbstractCommon wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

Download Full-text

Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

Journal of Fungi ◽

10.3390/jof7020149 ◽

2021 ◽

Vol 7 (2) ◽

pp. 149

Author(s):

Sarah-Maria Wege ◽

Katharina Gejer ◽

Fabienne Becker ◽

Michael Bölker ◽

Johannes Freitag ◽

...

Keyword(s):

Genome Editing ◽

Cell Biology ◽

Gene Deletion ◽

Fluorescent Protein ◽

Point Mutations ◽

Model Organism ◽

Ustilago Maydis ◽

Genomic Locus ◽

Targeted Gene Deletion ◽

Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

Download Full-text

Interdependent YpsA- and YfhS-Mediated Cell Division and Cell Size Phenotypes in Bacillus subtilis

mSphere ◽

10.1128/msphere.00655-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Robert S. Brzozowski ◽

Brooke R. Tomlinson ◽

Michael D. Sacco ◽

Judy J. Chen ◽

Anika N. Ali ◽

...

Keyword(s):

Bacillus Subtilis ◽

Cell Division ◽

Cell Size ◽

Unknown Function ◽

Suppressor Mutation ◽

Gram Positive ◽

Content Type ◽

Suppressor Mutations ◽

Extragenic Suppressor ◽

Cell Division Inhibition

ABSTRACT Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

Download Full-text