CRISPR-based engineering of next-generation lactic acid bacteria

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ScienceDirect CRISPR-based engineering of next-generation lactic acid bacteria Claudio Hidalgo-Cantabrana, Sarah O’Flaherty and Rodolphe Barrangou The advent of CRISPR-based technologies has opened new avenues for the development of next-generation food microorganisms and probiotics with enhanced functionalities. Building off two decades of functional genomics studies unraveling the genetic basis for food fermentations and host– probiotic interactions, CRISPR technologies offer a wide range of opportunities to engineer commercially-relevant Lactobacillus and Bifidobacteria. Endogenous CRISPR–Cas systems can be repurposed to enhance gene expression or provide new features to improve host colonization and promote human health. Alternatively, engineered CRISPR–Cas systems can be harnessed to genetically modify probiotics and enhance their therapeutic potential to deliver vaccines or modulate the host immune response. Address Department of Food, Bioprocessing and Nutritional Sciences, North Carolina State University, Raleigh, NC 27695, USA Corresponding author: Barrangou, Rodolphe ([email protected])

Current Opinion in Microbiology 2017, 37:79–87 This review comes from a themed issue on CRISPRcas9 Edited by David Bikard and Rodolphe Barrangou

http://dx.doi.org/10.1016/j.mib.2017.05.015 1369-5274/# 2017 Elsevier Ltd. All rights reserved.

Introduction Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated (Cas) proteins constitute adaptive immune systems against foreign nucleic acids such as phages [1] and plasmids [2]. These systems are present in many bacteria and most archaea, and constitute a diverse set of prokaryotic defense systems [3]. While the presence of CRISPR has been known since 1987 [4], their biological function was not elucidated until a decade ago, when studying phage resistance in the dairy starter culture Streptococcus thermophilus [5]. Although the majority of the scientific literature describes the use of CRISPR–Cas9 as a genetic engineering tool in eukaryotes, these systems hold tremendous potential for microbiology in general, and for the engineering of food cultures in particular [6]. Indeed, CRISPR–Cas systems www.sciencedirect.com

are widespread in lactic acid bacteria (LAB), bifidobacteria, and many members of the human microbiome [7– 9], as they confer a selective advantage against phages and plasmids. Type II CRISPR–Cas systems in particular are the most extensively studied to date, due to the genome editing capability of the programmable, precise, portable and efficient Cas9 signature nuclease. The Cas9 endonuclease can drive DNA binding and cleavage through an engineered single guide RNA (sgRNA) sequence [10]. This Cas9:sgRNA system has led to a wide variety of applications in human, plant, animal and microbe engineering, with the main focus on genome editing [11,12]. While using CRISPR for genome editing in eukaryotic systems has occurred at lightning speed due to the opportunity to cure human, animal and plant disease, relatively few studies have focused on bacterial genome editing, resulting in an arguably underutilized yet prodigious technology. Therefore, industrial microbes such as starter cultures and probiotic strains are a desirable target to harness CRISPR–Cas systems for genome engineering, especially given their important roles across the food supply chain, and their many biological functions spanning from food fermentation to human health promotion. In this regard, CRISPR–Cas systems will lead to improved probiotic features such as survival through gastrointestinal passage, host colonization, acid and bile resistance, and uptake and catabolism of non-digestible dietary oligosaccharides. Here, we discuss how genome engineering of LAB and bifidobacteria with CRISPR–Cas systems will improve industrially-relevant food bacteria, advance our understanding of their beneficial functionalities and enable engineering of strains for vaccine delivery and therapeutic purposes.

CRISPR–Cas systems to enhance starter cultures and probiotic strains Probiotics were originally defined as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ [13], a working definition that is continuously evolving [14] with recently published guidance for probiotic health claims [15]. Traditionally, several strains from select Lactobacillus and Bifidobacterium spp. have demonstrated a positive effect on human health and isolates from different species within these genera are widely used commercially as probiotic strains [16,17]. Moreover, with regards to fermented food products, these two genera together with S. thermophilus and Current Opinion in Microbiology 2017, 37:79–87

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Lactococcus lactis are the most commercially formulated starter cultures in the food industry. Conveniently, their frequent exposure to phage or foreign DNA in dairy environments, fermented foods and the gastrointestinal tract, has led to the occurrence of many native CRISPR– Cas systems in the genomes of LAB. Interestingly, a high percentage of Lactobacillus (62.9%) [18] and Bifidobacterium (77%) [19] encode CRISPR–Cas systems and remarkably 100 percentage of S. thermophilus strains do [20], although there are rarely present in L. lactis [21]. CRISPR–Cas systems comprise a variety of cas genes and repeat-spacer arrays, in which repeated sequences flank spacers that are short DNA sequences captured from invasive DNA during immunization. Spacers constitute a vaccination record and can be considered a genetic barcode for each strain. CRISPR systems are classified in different classes, types and subtypes, based on their signature cas genes [22] and on their mechanism of action (see contribution in this volume by Koonin et al.). The most widespread CRISPR–Cas systems belong to the Type I category, which accounts for 50% of all systems documented to date, whereas the popular Cas9 is derived from Type II systems. The plasticity of bacterial genomes, together with the ability to reprogram CRISPR–Cas systems is a potent technology to improve probiotic traits and starter culture functional characteristics through genome enhancement. Importantly, the broad occurrence of diverse CRISPR– Cas systems in LAB affords unique opportunities to repurpose these endogenous systems for various CRISPR-based applications. While several endogenous systems have been used for genotyping, they can also be repurposed for genome engineering applications, by delivering self-targeting templates on plasmids to alter genotypes, such as generating mutations, deletions or insertions by co-delivering recombination templates (Figure 1) [11]. To some extent, engineered native systems can also be repurposed for transcriptional control [23]. Repurposing endogenous systems also affords opportunities to by-pass the need to introduce foreign DNA, and opens intriguing avenues for non-GMO alternatives, notwithstanding ethical, legal and regulatory processes that must be updated in light of the CRISPR revolution underway. RNA and ribonucleoproteins (RNP) delivery certainly offer new regulatory avenues, notably, the use of preassembled CRISPR–Cas9 RNP loaded with guide RNAs [24]. Also, strains may be considered non-GMO when plasmids used for transformation are cured and no heterologous DNA remains. While CRISPR-based genome editing technologies have been adopted rapidly for eukaryotic applications, prokaryotic implementations have lagged behind. This is likely due to technical shortcomings such as transformation efficiency and the need to characterize and develop Current Opinion in Microbiology 2017, 37:79–87

new plasmids that can be used across a wide range of species and strains. Moreover, numerous bacterial species are still poorly characterized or uncultivable, and for those that are, many require improved growth conditions and genetic manipulation tools. Therefore, basic microbiological and molecular biology techniques must be advanced before CRISPR–Cas technologies are implemented. In instances in which CRISPR–Cas systems have been applied to bacteria as a genome-editing tool, the number of reports is minimal considering the potential of this technology (Table 1). These studies used CRISPR–Cas to perform site-specific mutations, gene insertions and deletions and for transcriptional regulation. To date, only a handful of these studies were performed in LAB (Table 1). In S. thermophilus, native CRISPR–Cas systems have been used as a screening tool to select specific genotypes naturally occurring in a heterogeneous population [25]. These systems can enable the selection for rare events that yield loss of single genes, or even the deletion of large, expendable genomic islands. CRISPR– Cas systems can likewise be used to modulate specific traits that enhance the starter culture phenotype such as exopolysaccharide (EPS) production [26]. For instance, a single nucleotide mutation could be efficiently introduced into polymerase epsC using the endogenous CRISPR–Cas system, in combination with a plasmid carrying a targeting guide RNA and a donor template sequence containing the mutation, resulting in the production of a different EPS with altered technological properties (Figure 1a), as shown in B. animalis subsp. lactis through classical molecular methods [27]. Modulating EPS synthesis would modify the rheological properties and enhance the organoleptic properties of dairy fermented products, in particular yoghurt and cheese. Moreover, genome enhancement in S. thermophilus based on their exposure to different phages could re-inforce their immune system against phages, ensuring perennial use of the best starter cultures. In Lactobacillus, numerous species harbor CRISPR–Cas systems in their genomes, with an unusually high frequency of Type II CRISPR– Cas systems [18]. In the commensal L. gasseri, several strains contain Type II systems perhaps reflecting widespread phage exposure, creating opportunities for the exploitation of endogenous systems for genome editing [28]. Likewise, Bifidobacterium spp. strains are often used as commercial probiotics, but have historically proven cumbersome to genetically manipulate through classical methods due to restriction-modification systems [29]. Several CRISPR–Cas systems have been identified in Bifidobacterium [19], though their activity is yet to be substantiated. Nevertheless, CRISPR–Cas technology would be an ideal means to genetically modify this genus, as to improve specific traits related to industrial manufacturing and human health enhancement, such as commercial processing, survival through gastrointestinal passage and immune-modulation. One key function to enhance is the ability of select Bifidobacterium species www.sciencedirect.com

LAB genome editings Hidalgo-Cantabrana, O’Flaherty and Barrangou 81

Figure 1

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(b)

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Current Opinion in Microbiology

Repurposing endogenous CRISPR–Cas systems to enhance lactic acid bacteria. Endogenous CRISPR–Cas9 systems can be re-directed by delivering plasmids that encode guide RNAs and template DNA for homologous recombination. The guide RNAs define the target sequence for Cas9, and trigger DNA repair pathways. (a) Alteration of the nucleotide sequence for epsC in Streptococcus thermophilus to enhance the production of exopolysaccharides, which are critical for dairy products texture. (b) Replacement of the bshA promoter sequence to increase the transcription of bshA, resulting in increased levels of bile salt hydrolysis by Lactobacillus gasseri. (c) Insertion of a novel carbohydrate hydrolase into Bifidobacterium longum subsp. longum, to enable the catabolism of a galacto-oligosaccharide, enhancing competitiveness for carbohydrate sources.

to catabolize non-digestible oligosaccharides, intensifying their ability to colonize the host and thrive on complex carbohydrates in the large intestine (Figure 1c). Bifidobacteria catabolize fructooligosaccharides (FOS) and bgalactooligosaccharides (GOS), but the efficiency is species or even strain dependent and also varies with the length of each oligosaccharide [30]. Recent work on human milk oligosaccharides (HMO) fermentation by bifidobacteria commonly found in breastmilk sets the stage for the enhancement of the catabolic potential of probiotics for improved ability to colonize the infant gut [31]. www.sciencedirect.com

Genetic modification of probiotic bacteria using traditional genetic systems has helped to understand, document and establish probiotic functionalities. Recently, the ability to perform targeted mutagenesis in the chromosome of a probiotic strain was demonstrated in Lactobacillus reuteri through CRISPR–Cas single-stranded DNA recombineering [32]. CRISPR–Cas systems have multiplexing potential, with the advantage that several deletions or genome manipulations can be performed concurrently. Therefore, CRISPR-based efforts are underway to enhance and understand probiotic function, ranging from acid and bile tolerance to manipulating the surface layer Current Opinion in Microbiology 2017, 37:79–87

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Table 1 CRISPR–Cas systems applied as genetic tools in bacteria. Purpose

Year

Reference

Site-specific mutation, gene insertion/deletion and transcriptional modulation Knockdown essential genes Gene insertion/deletion dCas9 to knockdown gene expression Gene insertion/deletion Plasmid targeting Gene insertion and deletion Targeted bacteria strain/species killing Function of native CRISPR Site-directed mutagenesis dCas9 to knockdown gene expression DNA targeting Gene insertion/deletion and transcriptional modulation Deletion of chromosomal targets

2016 2016 2016 2016 2016 2016 2015 2014 2015 2014 2017 2015 2015 2015

[60] [44] [61] [62] [63] [2] [64] [59] [28] [32] [44,45] [25] [65] [64]

Species Bacillus subtilis Clostridium beijerinckii Corynebacterium glutamicum Enterococcus faecalis Escherichia coli Lactobacillus gasseri a Lactobacillus reuteri a Staphylococcus aureus Streptococcus thermophilus a Streptomyces sp. Tatumella citrea a

Lactic acid bacteria.

such as the recently described surface layer associated proteins (SLAPs) [33,34]. For the former, enhancing bile salt hydrolase activity can help survival in the human gastrointestinal tract [35], and is useful to alter the pool of bile salts present in the intestine. As an example, endogenous CRISPR–Cas systems can be used to replace the original promoter of bshA with a highly efficient one (such as the pgm promoter), enhancing bshA transcription and boosting BshA activity (Figure 1b). For the latter, the cell surface of probiotic bacteria is an important area of research due to the intimate molecular interactions between SLAPs of bacteria and host epithelial cells. This molecular dialogue plays a role in the modulation of the host immune response, therefore the key proteins that mediate microbe–host interaction [36] are obvious targets for CRISPR–Cas genome engineering, such as fibronectin binding proteins (Fbp), mucin binding proteins (Mub), collagen binding proteins (Cbp) and yet to be characterized cell surface binding proteins [34]. Additional targets include traits that strengthen the strains to enhance their survival through gut transit, like EPS production [37], ability to store energy using the glycogen synthesis pathway [38] and H+ flux pumps [39]. Likewise, expression of adhesion and colonization structures like pili, transaldolase, enolase, or EPS could be increased to enhance host colonization and gut retention by probiotics [40,41] or even to modulate the host immune response [37]. Indeed, one of the main challenges for bacteria is their adaptation to new environments, and more specifically their capability to use complex carbohydrates sources [42]. Thus, genome enhancement of probiotic bacteria to impact promoters or to introduce carbohydrate uptake and catabolism metabolism genes will expand their carbon source options, increasing their competitiveness in complex ecological niche such as the human gut (Figure 1c). Genes encoding carbohydrate transporters and hydrolases involved in the uptake and catabolism of non-digestible oligosaccharides, respectively, could be Current Opinion in Microbiology 2017, 37:79–87

inserted into probiotic bacteria to enhance their competitiveness in the gut [43]. CRISPR–Cas genome editing is not restricted to the strains harboring endogenous CRISPR–Cas systems. When an endogenous system is not present, a CRISPR–Cas systems in their native (CRISPR–Cas9) or engineered (sgRNA:Cas9) form can be introduced on a plasmid with the required targeting guide RNAs for genome editing (Figure 2). Another option is to deliver a catalytically deactivated form of Cas9 (dCas9) that is able to bind the target DNA but not cleave it, representing a programmable DNA binding protein that blocks RNA polymerase and thus repress transcription [44,45,46]. Thereby, CRISPR-based technologies enable modulation of the transcriptome to study gene functionality in LAB and bifidobacteria (Figure 3). The multifunctional nature of Cas9, together with its programmable capability, allow the flexibility to use this system in bacteria that might contain CRISPR–Cas systems but not the endogenous CRISPR–Cas9 system. Thus, the construction of a plasmid harboring the two component Cas9:sgRNA system facilitates delivery of the genome editing machinery into target strains (Figure 2).

Cell surface engineering and vaccine delivery Regarding the genome editing possibilities in LAB and bifidobacteria, a key challenge will be to use these probiotic species for mucosal vaccine delivery to improve animal and human health. Mucosal vaccine delivery has been proposed as an alternative to traditional vaccines due to the reduction of secondary effects, ease of administration and modulation of mucosal immune and systemic responses [47]. Initially, Lactococcus lactis was the species of choice for heterologous expression due to the availability of plasmids, and its transformation potential [47]. Early work focused on expression of a protective antigen from Streptococcus mutans [48] and tetanus www.sciencedirect.com

LAB genome editings Hidalgo-Cantabrana, O’Flaherty and Barrangou 83

Figure 2

Ori

(a)

Ori

(b) Erm R

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E. coli

C. difficile Current Opinion in Microbiology

Delivering heterologous CRISPR–Cas9 systems for genome editing. A plasmid (pCas9) harboring the Cas9 nuclease, its single guide RNA, and a donor template for homologous recombination when applicable. (a) Insertion of an antigenic construct to alter the cell surface composition of Lactobacillus acidophilus, for vaccine delivery using oral probiotics. (b) Inactivation of tetW, a gene implicated in antibiotic resistance in Bifidobacterium animalis subsp. lactis. (c) Lethal self-targeting without a DNA repair template to selectively eliminate a bacterial genotype such as the spoilage organism Lactobacillus buchneri. This can also be exploited to target pathogenic bacteria.

fragment C [49,50]. In the following years, the use of Lactobacillus spp. superseded L. lactis for vaccine delivery due to their robustness, adjuvant properties and the generation of a wider range of genetic tools [51]. Traditionally, plasmids are used for transformation and expression of the desired antigen. However, antibiotic resistance genes as selection markers are undesirable due to the risk of horizontal transfer to commensal endogenous gut bacteria. Additionally, losing antigen expression in the absence of antibiotic pressure may occur, due to plasmid instability. Recombinant probiotic strains for mucosal vaccine delivery, harboring the genetic information in the chromosome, therefore lacking antibiotic resistance genes and plasmids, is arguably more desirable. This www.sciencedirect.com

strategy has been successfully used in LAB for delivery of bio-therapeutics [52] and antigens for vaccine expression in the probiotic strain Lactobacillus acidophilus NCFM, such as recent studies showing success against Clostridium botulinum and Bacillus anthracis [53,54]. Beyond providing the antigenic component, it is valuable to ensure there is a targeting peptide engineered into the construct to enable increased immune response, for instance by strategically targeting dendritic cells [55]. Utilizing CRISPR–Cas systems in LAB and bifidobacteria for rational design of vaccines and therapeutic strains will provide opportunities for precise genome engineering. As described above, the endogenous system can be Current Opinion in Microbiology 2017, 37:79–87

84 CRISPRcas9

Figure 3

Ori

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Ori

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Use of CRISPR-dCas9 variants for transcriptional regulation. The catalytically deactivated Cas9 (dCas9) can be delivered with a guide RNA to control transcription. (a) Transcription activation using dCas9 fused to a transcriptional activator to increase gene expression. (b) Transcriptional repression using dCas9 fused to a transcriptional repressor to block RNA polymerase activity.

harnessed or a Cas9:sgRNA system can be delivered to the probiotic strain for select DNA targeting (Figure 2). In this regard, the desired antigen will be cloned into the plasmid as a template donor with Cas9 complex (pCas9), and after Cas9-mediated cleavage, the donor sequence is used as a template to precisely insert the antigen in the bacterial chromosome (Figure 2a) via homologous recombination. Gene insertion could be performed into a highly constitutively expressed region or co-delivering a promoter to optimize the expression level of the vaccine. Finally, after genome editing is achieved, recombinant strains lack plasmids, thus reducing the potential risk of antibiotic resistance gene transfer. To date, bifidobacteria have not been used for mucosal vaccine delivery but this may change once CRISPR–Cas systems have been successfully used in this genus, opening up new avenues of research and applications. Actually, a major benefit of Current Opinion in Microbiology 2017, 37:79–87

CRISPR-driven genome editing will be the opportunity to readily alter the genomes of newly identified beneficial bacteria as microbiome efforts underway identify nextgeneration candidates, and bypass the need to tediously develop the basic tools to alter the genomes of new genera and species, which has historically been a substantial limitation. Regarding human health, the use of CRISPR technology to design vaccines for mucosal delivery could be applied to sexually transmitted diseases using members of the vaginal microbiota, namely Lactobacillus vaginalis, Lactobacillus jensenii, Lactobacillus gasseri or Lactobacillus crispatus [56]. In addition, CRISPR–Cas systems could be programmed to selectively target antibiotic resistance genes (or virulence genes) present in pathogens, in chromosomal or plasmid DNA, and screen for loss of antibiotic www.sciencedirect.com

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resistance genes, as recently shown in Staphylococcus aureus [45]. For instance, this application could be used to remove the tetW resistance gene, that is present in beneficial bacteria like B. animalis subsp. lactis (Figure 2b), improving time and efficiency compared with traditional molecular biology options [57]. Deleting antibiotic resistance genes from beneficial and pathogenic bacteria alike is desired as this will result in the reduction and transfer of antibiotic resistance, a main challenge for public health entities of developed countries. Finally, delivering Cas9 systems with various sgRNA enables the genesis of lethal chromosomal damage, leading to programmable bacteria death (Figure 2c). This could be exploited in food environments to selectively remove spoilage bacteria, such as L. buchneri, a spoilage microorganism in pickle fermentation [58]. This methodology may be also applied to the human microbiome to target pathogens such as Clostridium difficile, with unprecedented accuracy given the ability to distinguish even closely related genetic variants [59]. This approach could be used in combination with CRISPR-based antimicrobials (see other contribution in this volume by Bikard and Barrangou), enabling the alteration of complex ecological niche composition to improve human health. Thereof, human microbiome engineering and microbial community modulation are poised for implementation of CRISPR technologies, to remove or modulate the relative abundance of select species to improve human health.

Acknowledgements

The authors would like to thank their colleagues and support from NC State University, the North Carolina Ag Foundation and FEMS.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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Conclusions CRISPR–Cas systems afford intriguing new possibilities for genome enhancement of probiotic bacteria and food starter cultures to improve human health and safety in the food supply chain. Overall, CRISPR-based technologies are primed to be broadly implemented to enhance the functional attributes of existing industrial strains, and to develop next-generation food cultures with new properties. In addition to the functional benefits, CRISPRbased technologies are anticipated to shorten the product development cycle, increase the ease and efficiency with which alterations are carried out, and decrease time and costs associated with typical R&D efforts. Of course, common potential risks should always be considered when genetically modifying commercial strains and typical regulatory, ethical, and commercial consideration also apply. Nonetheless, the potential benefits are compelling and open new avenues for the genesis of improved probiotic strains to improve human health at un-precedented speed, ease and scale.

Conflicts of interest Rodolphe Barrangou is a co-inventor on several patents regarding CRISPR–Cas systems and their uses. RB is also a co-founder and SAB member of Intellia Therapeutics and Locus Biosciences. Claudio Hidalgo Cantabrana is on the scientific board and co-founder of Microviable Therapeutics. www.sciencedirect.com

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