SIBR-Cas enables host-independent and universal CRISPR genome engineering in bacteria

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and universal genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for universal and inducible control over CRISPR-Cas counterselection. This control delays CRISPR-Cas counterselection, granting more time for the editing event (e.g., by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three bacteria with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a universal gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.

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Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas

Nucleic Acids Research ◽

10.1093/nar/gkab893 ◽

2021 ◽

Author(s):

Constantinos Patinios ◽

Sjoerd C A Creutzburg ◽

Adini Q Arifah ◽

Belén Adiego-Pérez ◽

Evans A Gyimah ◽

...

Keyword(s):

Homologous Recombination ◽

Genome Engineering ◽

Wild Type ◽

Editing Event ◽

Pseudomonas Putida Kt2440 ◽

Knock Out ◽

Counter Selection ◽

Exogenous Expression ◽

Self Splicing ◽

Control Delays

Abstract CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria.

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

Microbial Cell Factories ◽

10.1186/s12934-019-1255-1 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 7

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.

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

10.1101/649921 ◽

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.

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Blocking drug efflux mechanisms facilitate genome engineering process in hypercellulolytic fungus, Penicillium funiculosum NCIM1228

Biotechnology for Biofuels ◽

10.1186/s13068-021-01883-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Anmoldeep Randhawa ◽

Nandita Pasari ◽

Tulika Sinha ◽

Mayank Gupta ◽

Anju M. Nair ◽

...

Keyword(s):

Homologous Recombination ◽

Genome Engineering ◽

Efflux Pump ◽

Genetic Manipulation ◽

Function Analysis ◽

Drug Tolerance ◽

Growth Media ◽

Penicillium Funiculosum ◽

Cellobiohydrolase I ◽

Drug Tolerability

Abstract Background Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods.

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Increasing the homologous recombination efficiency of eukaryotic microorganisms for enhanced genome engineering

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-09802-2 ◽

2019 ◽

Vol 103 (11) ◽

pp. 4313-4324 ◽

Cited By ~ 3

Author(s):

Ying Ding ◽

Kai-Feng Wang ◽

Wei-Jian Wang ◽

Yi-Rong Ma ◽

Tian-Qiong Shi ◽

...

Keyword(s):

Homologous Recombination ◽

Genome Engineering ◽

Recombination Efficiency ◽

Eukaryotic Microorganisms

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Impact of CRISPR-Cas9-Based Genome Engineering in Farm Animals

Veterinary Sciences ◽

10.3390/vetsci8070122 ◽

2021 ◽

Vol 8 (7) ◽

pp. 122

Author(s):

Parul Singh ◽

Syed Azmal Ali

Keyword(s):

Disease Resistance ◽

Genome Engineering ◽

Animal Health ◽

Fundamental Principle ◽

Farm Animals ◽

Web Based ◽

Knock Out ◽

Guide Rna ◽

Female Birth ◽

Comprehensive Information

Humans are sorely over-dependent on livestock for their daily basic need of food in the form of meat, milk, and eggs. Therefore, genetic engineering and transgenesis provide the opportunity for more significant gains and production in a short span of time. One of the best strategies is the genetic alteration of livestock to enhance the efficiency of food production (e.g., meat and milk), animal health, and welfare (animal population and disease). Moreover, genome engineering in the bovine is majorly focused on subjects such as disease resistance (e.g., tuberculosis), eradicate allergens (e.g., beta-lactoglobulin knock-out), products generation (e.g., meat from male and milk from female), male or female birth specifically (animal sexing), the introduction of valuable traits (e.g., stress tolerance and disease resistance) and their wellbeing (e.g., hornlessness). This review addressed the impressive genome engineering method CRISPR, its fundamental principle for generating highly efficient target-specific guide RNA, and the accompanying web-based tools. However, we have covered the remarkable roadmap of the CRISPR method from its conception to its use in cattle. Additionally, we have updated the comprehensive information on CRISPR-based gene editing in cattle.

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Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

Nature Communications ◽

10.1038/ncomms4045 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 28

Author(s):

Daniel Sommer ◽

Annika E. Peters ◽

Tristan Wirtz ◽

Maren Mai ◽

Justus Ackermann ◽

...

Keyword(s):

Homologous Recombination ◽

Genome Engineering ◽

Mouse Embryos ◽

Transcription Activator

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Analysis of relationship between key genes and astaxanthin biosynthesis in Phaffia rhodozyma by transcript level and gene knock-out

10.21203/rs.2.19181/v1 ◽

2019 ◽

Author(s):

Zhipeng Li ◽

Lina Chen ◽

Tianli Li ◽

Xiping Du ◽

Ning He ◽

...

Keyword(s):

Homologous Recombination ◽

Transcript Level ◽

Phaffia Rhodozyma ◽

Knock Out ◽

Astaxanthin Production ◽

Key Genes ◽

Different Strains ◽

The Relationship ◽

Rate Limiting ◽

Exact Relationship

Abstract Background Phaffia rhodozyma is a potential industrial source for production of natural astaxanthin. The synthetic mechanism of astaxanthin in P. rhodozyma is complex and unclear that blocked its development. Results In this study, eight genes related to dicyclic and monocyclic pathway in three different strains of P. rhodozyma were analyzed, and the relationship between the expression and astaxanthin biosynthesis was explored. Among these genes, crtYB (R=0.75, P<0.05) and asy genes (R=0.74, P<0.05) showed the most closely correlation with astaxanthin biosynthesis. In order to further study exact relationship, crtYB and asy genes were knocked out by homologous recombination. After crtYB knock-out, astaxanthin was decreased to be under detected line. It suggested crtYB played a role in dicyclic and monocyclic pathway. Meanwhile, the asy gene was in dicyclic pathway of astaxanthin biosynthesis, and its knock-out would promote the astaxanthin biosynthesis in monocyclic pathway, resulting in a 25.04% increase in astaxanthin production. Conclusion The possible rate-limiting enzymes were asy gene and crtYB illustrated by analysis of regression. Knock-out of asy and crtYB gene was great helpful to understand the synthetic pathway of astaxanthin, and significant to the industrial application of producing astaxanthin.

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Isolation of Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to Fosmidomycin

10.1101/296954 ◽

2018 ◽

Cited By ~ 1

Author(s):

Gur Pines ◽

Marcelo C. Bassalo ◽

Eun Joong Oh ◽

Alaksh Choudhury ◽

Andrew D. Garst ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Genome Engineering ◽

Acquired Resistance ◽

Specific Inhibitor ◽

Natural Process ◽

Future Generations ◽

Relative Fitness ◽

Mutant Library ◽

Acquired Drug Resistance ◽

E Coli

AbstractSequence to activity mapping technologies are rapidly developing, enabling the isolation of mutations that confer novel phenotypes. Here we used the CRISPR EnAbled Trackable genome Engineering (CREATE) technology to investigate the inhibition of the essential IspC gene in Escherichia coli. IspC gene product, Deoxyxylulose Phosphate Reductoisomerase (DXR), converts 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate in the DXP pathway. Since this pathway is shared with many pathogenic bacteria and protozoa and is missing in humans, it is an appealing target for inhibition. We created a full saturation library of 33 sites proximal to ligand binding and other sites and challenged it with the DXR-specific inhibitor, fosmidomycin. We identified several mutations that confer fosmidomycin resistance. All sites are highly conserved and also exist in pathogens including the malaria-inducing Plasmodium falciparum. These findings may have general implications on the isolation of resistance-conferring mutations and specifically, may affect the design of future generations of fosmidomycin-based drugs.SignificanceThe emergence of acquired drug resistance is a natural process that is likely to occur under most circumstances. Recently-developed technologies allow to map relative fitness contribution of multiple mutations in parallel. Such approaches may be used to predict which mutations are most likely to confer resistance, instead of waiting for them to evolve spontaneously. In this study, a rationally-designed IspC mutant library was generated genomically in E. coli. Mutants resistant to fosmidomycin, an antimalarial drug were identified, and most were in the highly conserved proline at position 274. These results may have implications on next-generation fosmidomycin drug design, and more broadly, this approach may be used for predicting mutational acquired resistance.

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Assembly of Radically Recoded E. coli Genome Segments

10.1101/070417 ◽

2016 ◽

Cited By ~ 1

Author(s):

Julie E. Norville ◽

Cameron L. Gardner ◽

Eduardo Aponte ◽

Conor K. Camplisson ◽

Alexandra Gonzales ◽

...

Keyword(s):

Homologous Recombination ◽

Genome Engineering ◽

Next Generation ◽

Selective Marker ◽

E Coli ◽

Cassette Exchange ◽

Large Potential ◽

Attachment Sites

AbstractThe large potential of radically recoded organisms (RROs) in medicine and industry depends on improved technologies for efficient assembly and testing of recoded genomes for biosafety and functionality. Here we describe a next generation platform for conjugative assembly genome engineering, termed CAGE 2.0, that enables the scarless integration of large synthetically recoded E. coli segments at isogenic and adjacent genomic loci. A stable tdk dual selective marker is employed to facilitate cyclical assembly and removal of attachment sites used for targeted segment delivery by sitespecific recombination. Bypassing the need for vector transformation harnesses the multi Mb capacity of CAGE, while minimizing artifacts associated with RecA-mediated homologous recombination. Our method expands the genome engineering toolkit for radical modification across many organisms and recombinase-mediated cassette exchange (RMCE).

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