Harnessing CRISPR-Cas systems for precision engineering of designer probiotic lactobacilli

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ScienceDirect Harnessing CRISPR-Cas systems for precision engineering of designer probiotic lactobacilli Yong Jun Goh and Rodolphe Barrangou Our evolving understanding on the mechanisms underlying the health-promoting attributes of probiotic lactobacilli, together with an expanding genome editing toolbox have made this genus an ideal chassis for the development of living therapeutics. The rising adoption of CRISPR-based technologies for prokaryotic engineering has demonstrated precise, efficient and scalable genome editing and tunable transcriptional regulation that can be translated into next-generation development of probiotic lactobacilli with enhanced robustness and designer functionalities. Here, we discuss how these tools in conjunction with the naturally abundant and diverse native CRISPR-Cas systems can be harnessed for Lactobacillus cell surface engineering and the delivery of biotherapeutics. Address Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Box 7624, Raleigh, NC 27695, United States Corresponding author: Barrangou, Rodolphe ([email protected])

Current Opinion in Biotechnology 2019, 56C:163–171 This review comes from a themed issue on Food biotechnology Edited by Rute Neves and Herwig Bachmann

https://doi.org/10.1016/j.copbio.2018.11.009 0958-1669/ã 2018 Elsevier Ltd. All rights reserved.

Introduction

´ lie First conceptualized by the Russian Nobel Laureate E Metchnikoff in 1906, the consumption of yogurt containing Lactobacillus bulgaricus enhances health and promotes longevity. This established a foundation for research interest on probiotics, defined as live microorganisms which, when administered in adequate amounts confer a health benefit on the host [1]. The genomics revolution has fueled probiotic research since the 1990s and continues to expand at an accelerated pace. Concurrently, our collective characterization and understanding of the link between the human microbiome and health in the past decade has proposed probiotic interventions as viable solutions to beneficially alter gut microbiome composition and address dysbiosis [2–6]. Beyond applications of allochthonous probiotics dominated by the genera Lactobacillus and Bifidobacterium in foods and as health www.sciencedirect.com

supplements, several species of Lactobacillus have also been exploited as chassis for mucosal delivery of vaccines and biotherapeutic agents [7,8] due to their record of safe use and unique adaptability to our gut environments. Another crucial milestone in probiotic research marked the development and continuous refinement of genetic tools for these otherwise intractable microorganisms to facilitate mechanistic studies as well as engineering of strain variants with tailored probiotic functionalities. Following the discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) in prokaryotes and the subsequent establishment of its adaptive immune function in the dairy bacterium Streptococcus thermophilus decades later [9–12], the advent of CRISPR-based technologies has catapulted the evolution of genome engineering across the tree of life. The sequence-specific targeting of nucleic acids via RNA-guided CRISPR-associated (Cas) effector proteins present unique opportunities for repurposing these defense machinery into programmable genome editing tools. In particular, the popular Cas9 endonuclease from the Streptococcus pyogenes (SpyCas9) Class 2 Type II CRISPR-Cas system, can be co-delivered with a target-specific chimeric single guide RNA (sgRNA) to drive precise DNA cleavage [13]. More recently, other Class 2 endonucleases, notably Cas12a (Cpf1), from Acidaminococcus and Lachnospiraceae, have also been exploited for genome editing applications, expanding the CRISPR toolbox by offering alternate sequence targeting requirements and cleavage pattern [14]. Gradually, CRISPRbased technologies are being implemented for genome editing in bacteria, including Escherichia coli, Streptococcus pneumoniae, species of Clostridium and Streptomyces, Lactococcus lactis, and probiotic Lactobacillus species [15,16,17,18,19,20]. The CRISPR genome engineering toolbox for lactobacilli can be further expanded by harnessing the naturally abundant endogenous CRISPR-Cas systems present within the genus, which can be reprogrammed for genome editing in the native hosts. Given the emerging roles of probiotic lactobacilli in foods and as living therapeutics, here we highlight how CRISPR-Cas systems can be harnessed for precise engineering of designer probiotics using endogenous Type I and II CRISPR-Cas systems, as well as by heterologous expression of Type II and V systems specifically in species or strains that do not possess functional CRISPR-Cas system.

CRISPR biology and the Cas toolbox To date, components of CRISPR-Cas systems were identified in approximately half of bacteria and the large Current Opinion in Biotechnology 2019, 56:163–171

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majority of archaea [21], reflecting the prevalence of this system in self-defense and regulation of genome plasticity in prokaryotes. Typically encoded in a genomic locus, the system is composed of CRISPR repeat-spacer arrays analogous to a genetic vaccination card, accompanied by a set of Cas proteins which altogether drive the DNAencoded, RNA-mediated, and nucleic acid targeting cell immunity in three distinct molecular stages: acquisition, expression, and interference. Acquisition occurs in the event of phage infection or invasive entry of exogenous nucleic acids, where specific Cas proteins capture a defined sequence of the foreign sequence element adjacent to specific sequence recognition pattern termed protospacer adjacent motif (PAM), and iteratively archive this ‘spacer’ into the CRISPR array. During expression, the CRISPR array is transcribed as a single pre-CRISPR RNA (crRNA) and processed into small interfering crRNAs each containing a spacer, followed by the interference stage in which the crRNAs guide the Cas effector nuclease for target-specific cleavage of complementary protospacer. Beyond RNA-guided adaptive immunity, an increasing body of evidence has also shed light into the moonlighting functions of CRISPR-Cas systems, including environmental sensing and gene regulation (reviewed in Refs. [22,23]). This reflects the central regulatory role of the Cas proteins which likely play critical roles in the persistence and probiotic functionalities of lactobacilli in their host environments. CRISPR-Cas systems are categorized into two evolutionary classes discerned by their signature Cas effector proteins, with multi-subunit effector complexes in Class 1 and single-protein effector modules in Class 2 systems [24]. The diversity of these systems was further highlighted by the subgrouping of these classes into 6 types (Type I, III and IV in Class 1; and II, V and VI in Class 2) and 29 subtypes identified to date, based on their genomic architecture, CRISPR repeats, Cas protein composition, effector complex structure, and the mechanisms of adaptation, crRNA processing and interference. Notably, most systems target DNA (i.e. Types I, II and V), and some systems target RNA (Types III and VI). The portability of single effector proteins from Class 2 systems such as the Cas9 signature nuclease from Type II systems and the Cas12 signature protein from Type V systems provides convenience for their heterologous expression on plasmids in a wide range of hosts along with self-targeting templates in the guide RNA to generate precise deletions, insertions or point mutations by codelivering a recombination template for editing. For all systems, the corresponding PAM sequences serve as license to cleavage by the Cas nucleases in the target DNA, and constitute an important criterion when designing target-specific guide RNAs. Due to the streamlined and portability of the Type II interference components, extensive research efforts have Current Opinion in Biotechnology 2019, 56:163–171

been invested into optimizing Cas9 and Cas12 and rational design of sgRNA to perform various editing (e.g. point mutations, gene knock-in/knock-out), transcriptional control and fluorescent imaging. Variants of Cas9 have been developed, such as Cas9 nickase (nCas9) with inactivated RuvC (Cas9D10A) or HNH (Cas9H840A) nuclease domain which results in single-stranded nick at the target DNA, thus greatly improving editing efficiencies in bacteria that lack repair mechanisms such as non-homologous end joining (NHEJ). Catalytically inactive variants of Cas9 (dCas9) and Cas12 (dCas12) with all nuclease domains inactivated and only binds to target DNA, has served as robust tools for gene silencing (CRISPRi) or tunable transcriptional regulation by interfering RNA polymerase activities at the promoter region or serving as a riboswitch [25]. To enhance base substitution functionality of Cas9 and Cas12, variants of these endonucleases have also been developed into efficient base editors (BEs) by fusing with a nucleoside deaminase, thereby improving base editing flexibility and specificity without double-stranded DNA cleavage [26,27].

Occurrence and distribution of CRISPR-Cas systems in probiotic lactobacilli Probiotic lactobacilli are among a diverse consortium of over 250 Lactobacillus species [28] belonging to the phylum Firmicutes. They are Gram-positive, non-spore-formers, G+C content ranging from 32 to 57% [29], predominantly fermentative, and produce lactic acid as the primary metabolic end product. With their saccharolytic nature, lactobacilli generally thrive in carbohydrate-rich environments, including milk, cereals, plants, and mucosal surfaces (oral, gastrointestinal [GI] tract and vaginal cavity) of mammals, reflecting their fastidious nutritional requirements. Historically as one of the most important groups of microbes used in food preservation, many species are considered as Generally Regarded As Safe (GRAS) and have long been associated with the production of fermented foods for which their desirable rapid acidification contributes to flavor and texture attributes and health-promoting properties. Of the >250 lactobacilli species, only about 20% have been consistently associated with the human gut [30], of which a small numbers are native (autochthonous) gut inhabitants while others are considered as transient (allochthonous), highlighting their specialized adaptation to the GI environment. Consequently, select strains of Lactobacillus, particularly those of human origin, have been exploited as probiotics. Owing to their primary active site in the small intestine despite at relative abundance of only 6% of the overall microbiota [31], lactobacilli play pivotal roles in modulating gut immunity and epithelial barrier function, colonization resistance against pathogens, the production or conversion of bioactive metabolites, digestion of dietary compounds and the regulation of host nutrient sensing and metabolism [32]. www.sciencedirect.com

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Presumably due to the ubiquitous nature of phages and mobile genetic elements (MGEs) in the native niches of lactobacilli species, elements of CRISPR-Cas systems are particularly enriched within the genus where CRISPR repeats were detected in 60% of 1262 lactobacilli genomes [33]. Indeed, most of the spacers in several species examined matched to phages and plasmids [33]. Distribution analysis of the complete CRISPR-Cas systems in lactobacilli revealed unusual overrepresentation of Type II (subtype A) systems, followed by Type I (subtype E) and III systems. No Type IV, V and VI systems were detected, although some lactobacilli appeared to have uncharacterized V-U proteins. For species commonly used as probiotics or are naturally associated with human host, most possess Type II systems, although some also encode Type I systems, with Lactobacillus fermentum and Lactobacillus salivarius possessing all three systems (Table 1). Notably, CRISPR-Cas systems are universal among strains of Lactobacillus jensenii and Lactobacillus mucosae; whereas all Lactobacillus acidophilus strains have degenerated CRISPR loci with only remnants of CRISPR arrays present in the genome. The enrichment of Type II systems in lactobacilli represents opportunities to exploit novel Cas9 proteins that have properties distinct from the canonical SpyCas9, SthCas9, SauCas9 or NmeCas9 for CRISPR editing with

expanded PAM specificities. Meanwhile, there is no report to date utilizing endogenous Type II CRISPR-Cas machinery for editing native bacterial genomes, whereas a limited number of studies have demonstrated the feasibility of genome editing using endogenous Type I systems [34,35]. This is largely due to the lack of NHEJ pathway in most prokaryotes for repair of double-stranded DNA break generated by Cas9, not to mention the inherently low homology-directed DNA repair efficiencies in bacteria. Nonetheless, considering heterologous SpyCas9 nickase variant has been employed successfully for genome editing in Lactobacillus casei [18], it is likely that the inactivation of one of the two nuclease domains in the native Cas9 will enable genome engineering applications with endogenous Type II systems in lactobacilli. The following section exemplifies the utility of endogenous Type I and II systems and heterologous Type II and V systems for engineering of probiotic lactobacilli as living therapeutics.

CRISPR-based engineering of tailored probiotics As aforementioned, implementation of CRISPR-Cas technologies in bacteria has increased, but remains surprisingly limited. CRISPR-based editing toolkits have already been developed for E. coli, B. subtilis, Clostridium,

Table 1 CRISPR-Cas systems in common probiotic species Species

No. of sequenced genomes

Genomes with cas genes (%)

Genomes with CRISPR repeats (%)

Type I (%)

Type II (%)

Type III (%)

Subtypes

L. acidophilus L. casei

16 36

0 75

100 75

0 19

0 61

0 0

L. crispatus

55

90

96

52

36

0

L. fermentum

32

75

78

37

43

9

L. gasseri

20

20

20

5

20

0

L. jensenii L. johnsonii

12 27

100 41

100 44

0 14

100 33

0 0

L. mucosae

6

100

100

33

33

0

L. plantarum

165

14

26

2

12

0

L. reuteri

82

18

17

0

8

0

L. rhamnosus L. salivarius

95 71

55 84

56 83

0 14

54 36

0 32

VU-4 I-E II-A, II-B, II-C I-B, I-E II-A, II-C VU-4 I-C, I-E II-A, II-B, II-C III-A VU-4 I-E II-A II-C I-E II-A, II-C VU-4 I-C, I-E II-C, VU-4 I-E II-A II-A VU-4 II-A I-E II-A III-A VU-4

Effector proteins: Type I, Cascade complex-Cas3; Type II, Cas9; Type III-A, Csm complex, Type VU-4, C2c9. Adapted from Crawley et al. [33].

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Corynebcterium and actinomycetes [16,36,37]. Among Lactobacillus species, Cas9-assisted single-stranded DNA recombineering was first performed in Lactobacillus reuteri for small deletions. More recently, Cas9-based gene deletions and insertion were performed in L. casei, and point mutations in Lactobacillus plantarum [17,18,19], with the latter work revealed variation in editing efficiencies based on strains and methods of repair template delivery [19]. In addition to the SpyCas9 toolbox, CRISPR editing tools based on the diverse repertoire of endogenous CRISPR-Cas systems in lactobacilli will undoubtedly facilitate the bioengineering of tailored probiotics approached from the perspectives of (a) enhancing the inherent properties of the microbes for improved stability and biodelivery, and (b) designing health and therapeutic functionalities utilizing probiotic microbes as the delivery chassis. Enhanced stress tolerance and gut fitness traits

One of the foremost criteria of a robust and effective probiotic is the ability to withstand industrial processing and maintain optimal performance in the host environment. For example, overexpression of the native molecular chaperone groESL or heterologous expression of a listerial betaine uptake system was demonstrated in Lactobacillus paracasei and Bifidobacterium breve to improve survival against temperature, oxidative and osmotic stresses during culture processing and enhanced gut persistence [38,39]. Other desirable probiotic properties that promote survival and retention in the host, such as the ability to synthesize glycogen as an energy storage compound [40], expression of putative cell surface adhesins including mucus-binding proteins [41,42], the production of exopolysaccharides (EPS) that mediate biofilm formation and enhance colonization in the gut, or increased expression of exporter systems for bile detoxification [43], can all potentially be modulated under in vivo conditions. In this case, CRISPR-based system can be used to replace for highly-inducible promoters to drive overexpression of these genes. The ability of probiotic lactobacilli to catabolize a broad range of dietary nondigestible carbohydrates or prebiotic oligosaccharide and host-derived glycans confer competitive advantage at the upper intestinal niche where these nutrient substrates are abundant. Most lactobacilli are endowed with a diverse classes of transporters and glycosyl hydrolases enabling them to utilize these substrates, although the overall carbohydrate utilization preference is species-specific and niche-specific. Some species of lactobacilli including L. acidophilus have also recently been shown to metabolize the glucoside moieties of certain dietary glycosylated phytochemicals and releasing the bioactive aglycone moieties, thus rendering these therapeutically active phytochemicals bioavailable to the host [44]. Using CRISPR-based gene knock-in strategy, these sugar catabolic genes can be introduced and expressed Current Opinion in Biotechnology 2019, 56:163–171

heterologously in another probiotic species, as exemplified by the expression of a L. paracasei cell surface associated beta-fructosidase in the established probiotic strain, Lactobacillus rhamnosus GG, thus expanding its metabolic capability for prebiotic fructooligosaccharides and other dietary nondigestible fructans [45]. HidalgoCantabrana et al. [46] recently provided a concise overview utilizing CRISPR-based editing approaches in probiotic lactobacilli and other food-relevant lactic acid bacteria to enhance functionalities, to drive the eradication of antibiotic-resistance genes and enable microbial community remodeling. Potential biotherapeutic delivery and prophylactic applications

Beyond L. lactis, E. coli and several other Gram-negative species, Lactobacillus strains have been increasingly favored as living therapeutic workhorses (reviewed in Refs. [47,48,49]) due to their inherent adjuvant properties and host immune tolerance, record of safe use, as well as their diverse niches that extend beyond the host GI tract and vaginal mucosal environment. Mucosal vaccines targeted against pathogens such as Bacillus anthracis, Clostridium botulinum, Clostridium difficile, pathogenic streptococci, and viruses such as rotavirus and HIV have been developed using lactobacilli as live vectors [7]. To circumvent the conventional genetic engineering method involving plasmid-encoded antigen delivery, marker-less gene replacement strategy was used for chromosomal expression of antigens in L. acidophilus from highly expressed loci driven by a strong constitutive promoter, or by fusion of viral epitope into the highly expressed surface layer (S-layer) proteins for explosive surface display of the antigen [50–52]. Recent microbiome studies have established Lactobacillus crispatus as one of the key indicators of a healthy vaginal microbiota [53]. For example, the endogenous Type I CRISPR-Cas systems in this species (Table 1) could be exploited for precise engineering of therapeutic strains for localized delivery of vaccines against sexually transmitted infections such as gonorrhea, HIV (Figure 1) and human papilloma virus (HPV). Likewise, Lactobacillus ultunensis or Lactobacillus gastricus, both commensal lactobacilli isolated from the stomach mucosa [54], are ideal candidates for potential vaccine delivery against Helicobacter pylori, an agent often associated with gastric ulcer and stomach cancer. Despite the general lack of CRISPR-Cas system in L. ultunensis, vaccine engineering of this species can be feasibly approached using heterologously expressed CRISPR systems such as the Cas12-based system (Figure 2). Other dimensions of lactobacilli-based therapeutics currently under development span from prophylactic control of inflammatory bowel disease (IBD), allergy and autoimmune diseases, and metabolic disorders (e.g. type I diabetes, obesity, and hypertension), to the delivery of anti-tumor agents and neurochemicals or psychoactive www.sciencedirect.com

CRISPR-based engineering of probiotic lactobacilli Goh and Barrangou 167

Figure 1

ori pLcri MPER

L. crispatus

S-layer proteins SlpA Cascade complex

cas3

pLgas F2RL1

L.gasseri L

tracrRNA

L

cas9

cas1 cas2

cas1 cas2 csn2 PAM

PAM protospacer

slpA

eno

cas9 cas3

cas1 cas2 csn2

cas1 cas2 crRNA crRNA repeat

repeat spacer

spacer Cascade+crRNA Cas9

Cas3

HDR PAR2 mimic

HDR

HIV-1 epitope

cas9 cas3

cas1 cas2 csn2

cas1 cas2 F2RL1

MPER

sIgA

IgG

Zot Z

V. cholera Current Opinion in Biotechnology

Reprogramming endogenous Type I-E and Type II-A CRISPR-Cas systems for targeted chromosomal insertions in vaginal-derived L. crispatus and gut commensal L. gasseri, respectively. For both endogenous systems, the (i) target-specific guide crRNA along with native leader sequence (L), and (ii) editing template composed of the desired insert sequence flanked by fragments homologous to the targeted insertion site, are delivered in plasmids into the hosts. For Type I system, the expressed crRNA directs target-specific binding of the Cascade and recruit Cas3 which nicks and progressively degrade the target complementary strand; whereas for Type II system, the crRNA:tracrRNA complex binds to Cas9 followed by double-stranded DNA cleavage by Cas9 at the target site. Homology-directed repair (HDR) by the hosts using the editing templates leads to precise insertion at the targeted sites in the recombinant strains. Left, L. crispatus expressing HIV-1 epitope for mucosal immunization, with the membrane proximal external region (MPER) of HIV-1 fused within the S-layer gene slpA for co-display on the cell surface [52]. Right, L. gasseri expressing human proteinase-activated receptor (PAR2) mimics which serve as binding decoy for Zonula occludens toxin (Zot) secreted by V. cholera. The PAR2-encoded gene, F2RL1 is inserted downstream of the highly expressed enolase gene as a single transcriptional unit.

compounds, the latter for the treatment of psychological disorders (reviewed in Refs. [47,48,55]). Cell surface components of lactobacilli such as the S-layers and lipoteichoic acids (LTA) are ligands for the host toll-like receptors (TLRs) and dendritic cells and thus elicit strain-dependent differential immunomodulation profiles in the host. In L. plantarum and L. acidophilus, modification of the LTA elicits anti-inflammatory response in murine colitis models. In addition, engineered L. www.sciencedirect.com

acidophilus deficient in LTA mitigated the formation of colonic cancer polyps in the ileum and colon of mouse model prone to developing colonic polyposis [56]. These studies demonstrated the therapeutic potentials of cell surface engineering for enhanced immunomodulatory properties of probiotic strains, which can be manipulated by programmable editing using CRISPR (Figure 2). Other live therapeutic approaches include exploiting the cell surface of lactobacilli as a scaffold for the display Current Opinion in Biotechnology 2019, 56:163–171

168 Food biotechnology

Figure 2

ncas9

ori

pLaci

L. acidophilus LTA

SlpA

cas12a pLult

Editing template

sgRNA

ureB

gRNA

L. ultunensis

pgm PAM

pgt

PAM

cas12a ncas9

ureB

Cas12a

HDR

nCas9

Urease B LTA-deficient

HDR

ureB

sIgA lumen urease

gut epithelium

H. pylori dendritic cell

IL-10 Current Opinion in Biotechnology

Heterologous expression of nCas9 and Cas12a (Cpf1) for genome editing in lactobacilli lacking a functional CRISPR-Cas system. Left, deletion of the phosphoglycerol transferase gene ( pgt) to disrupt LTA biosynthesis in the gut-associated L. acidophilus for enhanced anti-inflammatory properties. Right, chromosomal insertion of the H. pylori urease B gene (ureB) within a highly transcribed region in the gastric commensal L. ultunensis for cell surface expression of UreB and vaccination against H. pylori infection in the stomach mucosa. Expression of the plasmidencoded nCas9 or Cas12a along with the engineered gRNAs are driven by host-derived promoters cloned on the plasmids. Homology-directed repair (HDR) by the hosts mediated by the editing templates leads to precise gene insertion or deletion at the targeted sites in the recombinant strains.

of host receptor or toxin receptor mimics. The mimicry of host receptors acts as decoys for adhesion proteins or host oligosaccharide mimics present within the lipopolysaccharides of pathogens that have evolved to bind to host receptors, thereby interfering with ligand–receptor interactions between pathogens and the host. Other targets for receptor mimics engineered and under development include toxins produced by pathogenic E. coli, Vibrio cholera, and C. difficile (reviewed in Ref. [48]) which aimed to reduce symptoms of enteric infection. For example, the Zonula occludens toxin (Zot) produced Current Opinion in Biotechnology 2019, 56:163–171

by V. cholera interacts with host zonulin receptor leading to the disruption of intestinal tight junctions and mucosal barrier integrity [57]. In addition, dysregulation of host zonulin pathway has also been associated with the pathogenesis of IBD, celiac disease (CD) and type 1 diabetes [58]. As proposed in Figure 1, Lactobacillus gasseri, an autochthonous human gut commensal, possesses endogenous Type II CRISPR-Cas system which can be repurposed to express zonulin receptor mimic to chelate Zot and zonulin to ameliorate gut inflammation caused by compromised intestinal barrier function. An overall www.sciencedirect.com

CRISPR-based engineering of probiotic lactobacilli Goh and Barrangou 169

advantage of this decoy strategy is that there is no selective pressure on the pathogen to evolve their toxin affinity to the receptor mimics on the probiotic carrier [48].

Future perspectives Advances in CRISPR-based genome engineering and efforts underway to further democratize CRISPR tools and technologies have undoubtedly altered the landscape of genome editing from food to pharma, while illuminating the path for next-generation engineering of prokaryotic cell factories, including probiotic microbes for health and prophylactic applications. Integration of CRISPRbased editing with emerging synthetic biology approaches hold promises for the development of targeted therapeutic delivery along with rational design of programmable biological containment. While we anticipate the broad utility of the currently available Cas9 and Cas12 systems for engineering next-generation probiotics, the plethora of native CRISPR-Cas systems in lactobacilli constitutes an untapped resource for endogenous genome editing and remodeling, strain genotyping and vaccination against undesirable MGEs. This is in addition to the exploration of novel lactobacilli Cas9 and components of Type I and III systems as potentially valuable tools for expanding the current CRISPR toolbox with enhanced fidelity and specificities, and reduced immunogenic properties when delivered into mammalian systems. Overall, we are still at the infancy stage of developing personalized probiotics for precise and effective therapeutics in different host populations. With enhanced speed, efficiency and throughput, CRISPR-driven genome engineering builds upon the foundational genetic tools will leverage the research and development of lactobacilli-based true designer probiotics. Besides the technical aspects discussed here, it is important to also consider the regulatory issues related to the various uses of CRISPR-based technologies in general, and their applications in bacteria in particular. While several countries have elected not to regulate CRISPR-edited biological material (e.g. USDA-based endorsement of several Ag and food-based applications of the technology in 2018), others have already established broad barriers to their implementation in important markets (e.g. European Court of Justice deciding in 2018 that CRISPRbased technologies fall under the existing genetically modified organism guidelines). Moving forward, users must account for regulation-based limitations that may impact or even preclude the use of these technologies in various markets, and whether derived material is used as a food or a drug by consumers and patients, respectively.

Conflict of interest statement YJG and RB are inventors on several patents regarding probiotics and CRISPR-Cas systems and their uses. RB is also a co-founder and shareholder of Intellia Therapeutics and Locus Biosciences, and a shareholder of DuPont and Inari. www.sciencedirect.com

Acknowledgements The authors would like to acknowledge Rosemary Sanozky-Dawes for insightful discussion, and funding support from DuPont Nutrition & Health (USA) and the North Carolina Agricultural Foundation.

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