Chapter 17 : Genome Editing of Food-Grade Lactobacilli To Develop Therapeutic Probiotics

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For thousands of years, lactic acid bacteria (LAB) have been interwoven with our food supply. The earliest evidence of milk use dates back to 7,000 years BCE ( ), while cheeses were produced as early as 6,000 years BCE ( ). Advances in our understanding of the microbial world in the past couple of centuries have enabled microbiology-based food manufacturing on an industrial scale. Not only are LAB used to ferment dairy products, but they are also applied to pickle vegetables, to cure meats, and to produce alcoholic beverages such as wine and sake ( ). This long history of safe consumption led to the consideration that many LAB strains are Generally Recognized As Safe (GRAS). As early as 1906, LAB were linked to the promotion of human health. The Russian Nobel laureate Élie Metchnikoff hypothesized that ingestion of yogurt prolonged life in Eastern European populations by reducing “putrefying” [sic] bacteria. His linkage of the perceived longevity of the Eastern European populations with consumption of fermented dairy products ( ) made him the grandfather of modern probiotics. Probiotics are defined as “live microorganisms, which when administrated in adequate amounts, confer a health benefit to the host” ( ). Metchnikoff’s probiotic theory of life prolongation was never directly tested, and researchers reported in 1924 that LAB present in yogurt, specifically , most likely do not reduce “putrifying” bacteria in the intestine because did not survive gastrointestinal (GI) transit ( ). Other groups challenged this finding. Elli et al. demonstrated that both and were present in human feces after yogurt ingestion ( ).

Citation: van Pijkeren J, Barrangou R. 2018. Genome Editing of Food-Grade Lactobacilli To Develop Therapeutic Probiotics, p 389-408. In Britton R, Cani P (ed), Bugs as Drugs. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.BAD-0013-2016
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Figure 1

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).

Citation: van Pijkeren J, Barrangou R. 2018. Genome Editing of Food-Grade Lactobacilli To Develop Therapeutic Probiotics, p 389-408. In Britton R, Cani P (ed), Bugs as Drugs. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.BAD-0013-2016
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Image of Figure 2
Figure 2

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: (1) muramidase, also referred to as lysozyme, (2) glucosaminidase, (3) amidase, (4) γ-endopeptidase, and (5) endopeptidase. The figure is adapted from reference .

Citation: van Pijkeren J, Barrangou R. 2018. Genome Editing of Food-Grade Lactobacilli To Develop Therapeutic Probiotics, p 389-408. In Britton R, Cani P (ed), Bugs as Drugs. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.BAD-0013-2016
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Figure 3

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).

Citation: van Pijkeren J, Barrangou R. 2018. Genome Editing of Food-Grade Lactobacilli To Develop Therapeutic Probiotics, p 389-408. In Britton R, Cani P (ed), Bugs as Drugs. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.BAD-0013-2016
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Figure 4

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 ( ), 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.

Citation: van Pijkeren J, Barrangou R. 2018. Genome Editing of Food-Grade Lactobacilli To Develop Therapeutic Probiotics, p 389-408. In Britton R, Cani P (ed), Bugs as Drugs. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.BAD-0013-2016
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