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Genome Editing of Food-Grade Lactobacilli To Develop Therapeutic Probiotics

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  • Authors: Jan-Peter van Pijkeren1, Rodolphe Barrangou2
  • Editors: Robert Allen Britton3, Patrice D. Cani4
    Affiliations: 1: Department of Food Science, University of Wisconsin-Madison, Madison, WI 53706; 2: Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695; 3: Baylor College of Medicine, Houston, TX; 4: Université catholique de Louvain, Brussels, Belgium
  • Source: microbiolspec September 2017 vol. 5 no. 5 doi:10.1128/microbiolspec.BAD-0013-2016
  • Received 23 January 2017 Accepted 21 February 2017 Published 29 September 2017
  • Jan-Peter van Pijkeren, [email protected]
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  • Abstract:

    Lactic acid bacteria have been used historically for food manufacturing mainly to ensure preservation via fermentation. More recently, lactic acid bacteria have been exploited to promote human health, and many strains serve as industrial workhorses. Recent advances in microbiology and molecular biology have contributed to understanding the genetic basis of many of their functional attributes. These include dissection of biochemical processes that drive food fermentation, and identification and characterization of health-promoting features that positively impact the composition and roles of microbiomes in human health. Recently, the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-based technologies has revolutionized our ability to manipulate genomes, and we are on the cusp of a broad-scale genome editing revolution. Here, we discuss recent advances in genetic alteration of food-grade bacteria, with a focus on CRISPR-associated enzyme genome editing, single-stranded DNA recombineering, and the modification of bacteriophages. These tools open new avenues for the genesis of next-generation biotherapeutic agents with improved genotypes and enhanced health-promoting functional features.

  • Citation: van Pijkeren J, Barrangou R. 2017. Genome Editing of Food-Grade Lactobacilli To Develop Therapeutic Probiotics. Microbiol Spectrum 5(5):BAD-0013-2016. doi:10.1128/microbiolspec.BAD-0013-2016.


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Lactic acid bacteria have been used historically for food manufacturing mainly to ensure preservation via fermentation. More recently, lactic acid bacteria have been exploited to promote human health, and many strains serve as industrial workhorses. Recent advances in microbiology and molecular biology have contributed to understanding the genetic basis of many of their functional attributes. These include dissection of biochemical processes that drive food fermentation, and identification and characterization of health-promoting features that positively impact the composition and roles of microbiomes in human health. Recently, the advent of clustered regularly interspaced short palindromic repeat (CRISPR)-based technologies has revolutionized our ability to manipulate genomes, and we are on the cusp of a broad-scale genome editing revolution. Here, we discuss recent advances in genetic alteration of food-grade bacteria, with a focus on CRISPR-associated enzyme genome editing, single-stranded DNA recombineering, and the modification of bacteriophages. These tools open new avenues for the genesis of next-generation biotherapeutic agents with improved genotypes and enhanced health-promoting functional features.

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CRISPR-Cas systems. Two primary classes of CRISPR-Cas systems have been established, based on the nature of the effector proteins that direct targeting: either multisubunit complexes (class 1) or single effector proteins (class 2). Each major type of effector protein drives select cleavage of target nucleic acid, generating single-strand exonucleolytic cleavage (type I), shredding (type III), unknown (type IV), blunt cleavage (types II and VI), or sticky-end dual nicking (type V).

Source: microbiolspec September 2017 vol. 5 no. 5 doi:10.1128/microbiolspec.BAD-0013-2016
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Endolysin target sites within the Gram-positive peptidoglycan matrix. A simplified overview of the peptidoglycan matrix in which the target sites of the five bacteriophage-derived endolysins are indicated with green arrows. The arrows refer to the following endolysin types: muramidase, also referred to as lysozyme, glucosaminidase, amidase, γ-endopeptidase, and endopeptidase. The figure is adapted from reference 120 .

Source: microbiolspec September 2017 vol. 5 no. 5 doi:10.1128/microbiolspec.BAD-0013-2016
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Repurposing CRISPR-Cas systems as antimicrobials. If endogenous CRISPR-Cas systems are natively present in the target organism (left), they can be repurposed and redirected toward self-targeting by delivering either CRISPR guide RNAs or synthetic CRISPR arrays that contain a self-targeting spacer that contains sequences homologous to those of the host chromosome. Alternatively, for organisms in which no CRISPR-Cas systems are universally present, or active (right), both the CRISPR arrays (or guide RNAs) and the Cas machinery (Cas effector nucleases such as Cascade or Cas9) can be delivered via plasmids or phages. Various types of CRISPR-Cas systems can be harnessed for lethal self-targeting (bottom), encompassing both class 1 and class 2 systems, exemplified by the type I-E system, hinging on the Cas3 exonuclease for extensive shredding of a DNA strand (bottom left), or by the type II-A system, hinging on the Cas9 endonuclease for genesis of double-stranded DNA breaks (bottom right).

Source: microbiolspec September 2017 vol. 5 no. 5 doi:10.1128/microbiolspec.BAD-0013-2016
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Probiotic dual-delivery system of CRISPR-coding bacteriophages. Conceptual overview of an engineered probiotic encoding phasmid-derived virions that harbor a CRISPR array to target pathogens upon release from the probiotic delivery host. Amplicons of a pathogen-derived double-stranded DNA bacteriophage are fused with a plasmid origin of replication (ORI), a probiotic auxotrophic marker, and a CRISPR cassette. The phasmid-encoded auxotrophic marker, when deleted from the bacterial chromosome, yields stable phasmid replication. The phasmid will reproduce virions, which encode engineered CRISPR arrays, in the cytosol of the cell. Release of the engineered virions can be achieved by placing a gene encoding a holin and/or endolysin protein, which is known to lyse the probiotic, under the control of a promoter that is activated upon sensing environmental cues, i.e., bile salts, in the small intestine. These already have been identified in bacteria ( 156 ), which can be adapted for use in probiotics. Successful lysis achieves both biological containment and delivery of the engineered virions . When the virions attach to the target pathogen, DNA will be injected. Delivery of the user-defined CRISPR array will, combined with native Cas enzymes, result in strain-specific killing of the pathogen.

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