scholarly journals Inducible Plasmid Self-Destruction (IPSD) assisted genome engineering in lactobacilli and bifidobacteria

2019 ◽  
Author(s):  
Fanglei Zuo ◽  
Zhu Zeng ◽  
Lennart Hammarström ◽  
Harold Marcotte
Keyword(s):  
Synthetic Biology ◽  
Homologous Recombination ◽  
Genetic Engineering ◽  
Genome Editing ◽  
Genome Engineering ◽  
Bacterial Species ◽  
Knock Out ◽  
Recombination System ◽  
A Genome ◽  
Selection Of

ABSTRACTGenome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria but still, have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called Inducible Plasmid Self-Destruction (IPSD) was successfully used to perform gene knock-out and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.

scholarly journals Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Ioannis Mougiakos ◽  
Enrico Orsi ◽  
Mohammad Rifqi Ghiffary ◽  
Wilbert Post ◽  
Alberto de Maria ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Homologous Recombination ◽  
Genome Editing ◽  
Rhodobacter Sphaeroides ◽  
Genome Engineering ◽  
Start Codon ◽  
Terpene Biosynthesis ◽  
Knock Out ◽  
A Genome ◽  
Coa Reductase

Abstract Background Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides. Results Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected. Conclusions In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

scholarly journals Development of oriC-Based Plasmids for Mesoplasma florum

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Dominick Matteau ◽  
Marie-Eve Pepin ◽  
Vincent Baby ◽  
Samuel Gauthier ◽  
Mélissa Arango Giraldo ◽  
...  
Keyword(s):  
Systems Biology ◽  
Synthetic Biology ◽  
Genetic Engineering ◽  
Genome Engineering ◽  
Antibiotic Resistance Genes ◽  
Viable Cell ◽  
Pathogenic Potential ◽  
Strong Basis ◽  
Content Type ◽  
Basic Biology

ABSTRACT The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10−6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10−6 and 8.44 × 10−7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum. Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology. IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering

2016 ◽  
Vol 45 (24) ◽  
pp. 6666-6684 ◽  
Author(s):  
Amrita Singh ◽  
Debojyoti Chakraborty ◽  
Souvik Maiti
Keyword(s):  
Genome Editing ◽  
Chemical Biology ◽  
Genome Engineering ◽  
A Genome

The development and adaptation of CRISPR–Cas9 as a genome editing tool and chemical biology approaches for modulating its activity.

scholarly journals CRISPR-Cas9 System for Plant Genome Editing: Current Approaches and Emerging Developments

Agronomy ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1033 ◽  
Author(s):  
Jake Adolf V. Montecillo ◽  
Luan Luong Chu ◽  
Hanhong Bae
Keyword(s):  
Genetic Engineering ◽  
Genome Editing ◽  
Gene Editing ◽  
Fine Tuning ◽  
Plant Genome ◽  
Detection Methods ◽  
Editing Efficiency ◽  
Targeted Gene Editing ◽  
Transgene Detection ◽  
Selection Of

Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

scholarly journals Base editors: modular tools for the introduction of point mutations in living cells

2019 ◽  
Vol 3 (5) ◽  
pp. 483-491 ◽  
Author(s):  
Mallory Evanoff ◽  
Alexis C. Komor
Keyword(s):  
Synthetic Biology ◽  
Genome Editing ◽  
Genome Engineering ◽  
Point Mutations ◽  
Living Cells ◽  
Single Base ◽  
New Family ◽  
Alternative Techniques ◽  
Cas Proteins ◽  
Modular Nature

Base editors are a new family of programmable genome editing tools that fuse ssDNA (single-stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.

scholarly journals Highly efficient generation of bacteria leaf blight resistance and transgene-free rice using genome editing and multiplexed selection system

2020 ◽  
Author(s):  
Kun Yu ◽  
Zhiqiang Liu ◽  
Huaping Gui ◽  
Lizhao Geng ◽  
Juan Wei ◽  
...  
Keyword(s):  
Protein Binding ◽  
Genome Editing ◽  
Leaf Blight ◽  
Genomic Research ◽  
Blight Resistance ◽  
Selection System ◽  
Transcription Activator ◽  
Efficient Generation ◽  
A Genome ◽  
Selection Of

Abstract Background Rice leaf blight is a worldwide devastating disease caused by bacteria Xanthomonas oryzae pv. Oryzae (Xoo). The UPT (up-regulated by transcription activator-like 1 effector) box in promoter region of the rice Xa13 gene played a key role in Xoo pathogenicity. Mutation of key bacterial protein binding site in UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistant to bacterial.Highly efficient generation and selection transgene-free, edited plants helpful to shorten and simple the gene editing breeding process. Selective elimination of transgenic pollen of E0 plants can enrich proportion of E1 transgene-free offspring and expression of the color mark gene in seeds makes the selection of E2 plants is very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacteria leaf blight resistance and transgene-free rice plants.Results We introduced site specific mutations into the UPT box using CRISPR/Cas12a technology to hamper TAL (Transcription-Activator Like effectors) protein binding and gene activation, and generated genome edited rice with improved bacteria blight resistance. Transgenic pollens of E0 plants were eliminated by pollen specific expression of α-amylase gene Zmaa1, the proportion of transgene-free plants were enriched from 25% to 50% in single T-DNA insertion events in E1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced 50% cost and achieved up to 98.64% of accuracy for selection of transgene-free edited plants. Conclusion We demonstrated core nucleotide deletion in the UPT box of Xa13 promoter conferred resistance to rice blight and selection of transgene-free plants were boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

scholarly journals Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Wei Shen ◽  
Jun Zhang ◽  
Binan Geng ◽  
Mengyue Qiu ◽  
Mimi Hu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Zymomonas Mobilis ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Strain Improvement ◽  
Genomic Research ◽  
Francisella Novicida ◽  
A Genome

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

scholarly journals CRISPR/Cas Technologies and Their Applications in Escherichia coli

2021 ◽  
Vol 9 ◽  
Author(s):  
Huina Dong ◽  
Yali Cui ◽  
Dawei Zhang
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Regulation ◽  
Homologous Recombination ◽  
Genome Editing ◽  
State Of The Art ◽  
Bacterial Species ◽  
The State ◽  
Research Progress ◽  
End Joining ◽  
Eukaryotic Organism

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have revolutionized genome editing and greatly promoted the development of biotechnology. However, these systems unfortunately have not been developed and applied in bacteria as extensively as in eukaryotic organism. Here, the research progress on the most widely used CRISPR/Cas tools and their applications in Escherichia coli is summarized. Genome editing based on homologous recombination, non-homologous DNA end-joining, transposons, and base editors are discussed. Finally, the state of the art of transcriptional regulation using CRISPRi is briefly reviewed. This review provides a useful reference for the application of CRISPR/Cas systems in other bacterial species.

scholarly journals Multiplex genomic edition in Ashbya gossypii using CRISPR-Cpf1

2019 ◽  
Author(s):  
Alberto Jiménez ◽  
Birgit Hoff ◽  
José Luis Revuelta
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Marker Genes ◽  
Target Sequence ◽  
Ashbya Gossypii ◽  
Nucleotide Substitutions ◽  
Large Deletions ◽  
Auxotrophic Marker ◽  
Selection Of

AbstractThe CRISPR/Cas technologies constitute an essential tool for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables the efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained to the existence of an specific 5’-NGG-3’ PAM sequence in the target site.Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows to employ a T-rich PAM sequence (5’-TTTN-3’) and facilitates the implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions into five different auxotrophic marker genes (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system was demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multi-CRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may significantly affect to the edition efficiency of the system.

scholarly journals Efficient CRISPR/Cas12a-based genome editing toolbox for metabolic engineering in Methanococcus maripaludis

2021 ◽  
Author(s):  
Jichen Bao ◽  
Silvan Scheller
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Value Added ◽  
Primary Metabolism ◽  
Methanococcus Maripaludis ◽  
Knock Out ◽  
Homology Directed Repair ◽  
A Genome ◽  
Product Scope ◽  
Positive Rate

Methanococcus maripaludis is a fast-growing and genetically tractable methanogen. To become a useful host organism for the biotechnological conversion of CO2 and renewable hydrogen to fuels and value-added products, its product scope needs to be extended. Metabolic engineering requires reliable and efficient genetic tools, in particular for genome editing related to the primary metabolism that may affect cell growth. We have constructed a genome editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery natively present in M. maripaludis. The toolbox enables gene knock-out with a positive rate typically above 89%, despite M. maripaludis being hyper-polyploid. We have replaced the flagellum operon (around 8.9kb) by a beta-glucuronidase gene to demonstrate a larger deletion, and to enable quantification of promotor strengths. The CRISPR/LbCas12a toolbox presented here is currently perhaps the most reliable and fastest method for genome editing in a methanogen.

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