Abstract
Human body is colonized by a complex ecosystem of microbes that play a significant role in determining the fitness of the host. The epigenetic signatures of probiotic microorganisms also define the functional role of microbiota in the intestine and further facilitate exploring their beneficial properties. Development in the areas of molecular biology and genomics has paved way for engineering of microorganisms. The advent of genome editing tools such as CRISPR-Cas, has revolutionized new possibilities for genetic engineering in probiotics for enhanced immune response, antimicrobial properties and applications in biotherapeutics. Here, we discuss the present status of genome editing used in epigenome engineering of the gut microbiota and role of different types of CRISPR-Cas systems. Applications of CRISPR-Cas system can be harnessed to modify probiotic microbes in order to regulate gene expressions and produce desired metabolite. These techniques illuminate novel approaches to metabolic engineering and draw attention towards poorly characterized biosynthetic pathways. In addition, future prospects of these tools can be applied to investigate the interactions between gut microbiome and the host, hence, contributing to the development of novel therapeutics.
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Abbreviations
- LAB:
-
Lactic Acid Bacteria
- DSB:
-
Double Strand Break
- HR:
-
Homologous Recombination
- NHEJ:
-
Non-Homologous End Joining
- pyrE:
-
Phosphoribosyltransferase
- HDR:
-
Homology Directed Repair
- crRNA:
-
CRISPR RNA
- gRNA:
-
Guide RNA
- IPSD:
-
Inducible Plasmid Self-Destruction
- MDR:
-
Multidrug-Resistant
- AMP:
-
Antimicrobial Peptides
- Lc. lactis :
-
Lactococcus lactis
- AMR:
-
Antimicrobial-Resistant
- CRISPRi:
-
CRISPR interference
- dCas9:
-
Dead Cas9
- GFP:
-
Green Fluorescent Protein
- CRISPRa:
-
CRISPR activation
- IBD:
-
Inflammatory bowel disease
- UC:
-
Ulcerative colitis
- Gatm :
-
Glycine Aminotransferase
- S-layers:
-
Surface Layers
- Slps:
-
S-layer proteins
- Lb. crispatus :
-
Lactobacillus crispatus
- B. longum :
-
Bifidobacterium longum
- S. cerevisiae :
-
Saccharomyces cerevisiae
- S. boulardii :
-
Saccharomyces boulardii
- GRAS:
-
Generally Recognized As Safe
References
Azad, Md. A. K., Sarker, M., Li, T., & Yin, J. (2018). Probiotic species in the modulation of gut microbiota: An overview. Biomed Res Int.1–8. https://doi.org/10.1155/2018/9478630
Bermúdez-Humarán LG, Motta JP, Aubry C, Kharrat P, Rous-Martin L, Sallenave JM, Deraison C, Vergnolle N, Langella P (2015) Serine protease inhibitors protect better than IL-10 and TGF-β anti-inflammatory cytokines against mouse colitis when delivered by recombinant lactococci. Microb Cell Fact 14:26. https://doi.org/10.1186/s12934-015-0198-4
Berlec A, Škrlec K, Kocjan J, Olenic M, Štrukelj B (2018) Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis. Sci Rep 8(1):1–11. https://doi.org/10.1038/s41598-018-19402-1
Bibikova M (2003) Enhancing Gene Targeting with Designed Zinc Finger Nucleases. Science 300(5620):764–764. https://doi.org/10.1126/science.1079512
Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, Fischetti VA, Marraffini LA (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 32:1146–1150. https://doi.org/10.1038/nbt.3043
Cano-Garrido, O., Seras-Franzoso, J., & Garcia-Fruitós, E. (2015). Lactic acid bacteria: reviewing the potential of a promising delivery live vector for biomedical purposes. Microb Cell Fact. 14. https://doi.org/10.1186/s12934-015-0313-6
Chakraborty AK, Muneim GE, Pradhan S, Adhikari A (2018) Superbug horror and its relations to antibiotics, probiotics and vitamins. J Pharm Toxicol. 1(1):14–19
Citorik RJ, Mimee M, Lu TK (2014) Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol 32:1141–1145. https://doi.org/10.1038/nbt.3011
Clavel T, Haller D (2007) Bacteria- and host-derived mechanisms to control intestinal epithelial cell homeostasis: Implications for chronic inflammation. Inflamm Bowel Dis 13(9):1153–1164. https://doi.org/10.1002/ibd.20174
Cordaillat-Simmons, M., Rouanet, A., & Pot, B. (2020). Live biotherapeutic products: the importance of a defined regulatory framework. Exp Mol Med, 1–10. https://doi.org/10.1038/s12276-020-0437-6
Crawley A, Henriksen E, Stout E, Brandt K, Barrangou R (2018) Characterizing the activity of abundant, diverse and active CRISPR-Cas systems in lactobacilli. Sci Rep 8:11544. https://doi.org/10.1038/s41598-018-29746-3
Deveau H, Garneau JE, Moineau S (2010) CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol 64(1):475–493. https://doi.org/10.1146/annurev.micro.112408.134123
Dong C, Fontana J, Patel A, Carothers JM, Zalatan JG (2018) Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria. Nat Comm 9(1):2489. https://doi.org/10.1038/s41467-018-04901-6
Dotto GP, Enea V, Zinder ND (1981) Functional analysis of bacteriophage f1 intergenic region. Virol J 114:463–473. https://doi.org/10.1016/0042-6822(81)90226-9
Duan FF, Liu JH, March JC (2015) Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. J Diabetes 64(5):1794–1803. https://doi.org/10.2337/db14-0635
Fagan RP, Fairweather NF (2014) Biogenesis and functions of bacterial S-layers. Nat Rev Microbiol 12:211–222. https://doi.org/10.1038/nrmicro3213
Goh, Y. J., Azcárate-Peril, M. A., O'Flaherty, S., Durmaz, E., Valence, F., Jardin, J., Lortal, S., & Klaenhammer, T. R. (2009). Development and application of a upp-based counterselective gene replacement system for the study of the S-layer protein SlpX of Lactobacillus acidophilus NCFM. Appl Environ Microbiol, 75(10), 3093–3105. https://doi.org/10.1128/AEM.02502-08
Hidalgo-Cantabrana C, Goh YJ, Pan M, Sanozky-Dawes R, Barrangou R (2019) Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus. PNAS 116(32):15774–15783. https://doi.org/10.1073/pnas.1905421116
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278. https://doi.org/10.1016/j.cell.2014.05.010
Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR (2018) Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556(7699):57–63. https://doi.org/10.1038/nature26155
Hynonen U, Palva A (2013) Lactobacillus surface layer proteins: structure, function and applications. Appl Microbiol Biotechnol 97:5225–5243. https://doi.org/10.1007/s00253-013-4962-2
Jaimee G, Halami PM (2016) Emerging resistance to aminoglycosides in lactic acid bacteria of food origin-an impending menace. Appl Microbiol Biotechnol 100(3):1137–1151. https://doi.org/10.1007/s00253-015-7184-y
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 32:1146–1150. https://doi.org/10.1038/nbt.2508
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. https://doi.org/10.1126/science.1225829
Kerry RG, Patra JK, Gouda S, Park Y, Shin H-S, Das G (2018) Benefaction of probiotics for human health: A review. J Food Drug Anal 26(3):927–939. https://doi.org/10.1016/j.jfda.2018.01.002
Kim SR, Kwee NR, Kim H, Jin Y-S (2013) Feasibility of xylose fermentation by engineered Saccharomyces cerevisiae overexpressing endogenous aldose reductase (GRE3), xylitol dehydrogenase (XYL2), and xylulokinase (XYL3) from Scheffersomyces stipitis. FEMS Yeast Res 13(3):312–321. https://doi.org/10.1111/1567-1364.12036
Klotz C, Barrangou R (2018) Engineering components of the lactobacillus S-layer for biotherapeutic applications. Front Microbiol 9:2264. https://doi.org/10.3389/fmicb.2018.02264
Kommineni S, Bretl DJ, Lam V, Chakraborty R, Hayward M, Simpson P, Cao Y, Bousounis P, Kristich CJ, Salzman NH (2015) Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract. Nature 526(7575):719–722. https://doi.org/10.1038/nature15524
Kumar P, Kizhakkedathu JN, Straus SK (2018) Antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity and biocompatibility in vivo”. Biomolecules 8(1):4. https://doi.org/10.3390/biom8010004
Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS (2013) CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc 8(11):2180–2196. https://doi.org/10.1038/nprot.2013.132
Lee JH, O’Sullivan DJ (2008) Metabolic engineering of Lactococcus lactis for the development of a one-step bioconversion of lactose into tagatose. ASM Annual Meeting, Boston, MA
Lee SY, Park JM, Kim TY (2011) Application of metabolic flux analysis in metabolic engineering. Meth Enzymol 498:67–93. https://doi.org/10.1016/B978-0-12-385120-8.00004-8
Leenay RT, Vento JM, Shah M, Martino ME, Leulier F, Beisel CL (2018) Streamlined, recombinase-free genome editing with CRISPR-Cas9 in Lactobacillus plantarum reveals barriers to efficient editing [Preprint]. ACS Synth Biol. https://doi.org/10.1101/352039
Leonard SP, Perutka J, Powell JE, Geng P, Richhart DD, Byrom M, Kar S, Davies BW, Ellington AD, Moran NA, Barrick JE (2018) Genetic engineering of bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids. ACS Synth Biol 7(5):1279–1290. https://doi.org/10.1021/acssynbio.7b00399
Li B, Liu F, Tang Y, Luo G, Evivie S, Zhang D, Wang N, Li W, Huo G (2015) Complete genome sequence of Lactobacillus helveticus KLDS1.8701, a probiotic strain producing bacteriocin. J Biotechnol 212:90–91. https://doi.org/10.1016/j.jbiotec.2015.08.014
Limanskiy V, Vyas A, Chaturvedi LS, Vyas D (2019) Harnessing the potential of gene editing technology using CRISPR in inflammatory bowel disease. World J Gastroenterol 25(18):2177–2187. https://doi.org/10.3748/wjg.v25.i18.2177
Liu J-J, Kong II, Zhang G-C, Jayakody LN, Kim H, Xia P-F, Kwak S, Sung BH, Sohn J-H, Walukiewicz HE, Rao CV, Jin Y-S (2016) Metabolic engineering of probiotic Saccharomyces boulardii. Appl Environ Microbiol 82(8):2280–2287. https://doi.org/10.1128/AEM.00057-16
Luo ML, Mullis AS, Leenay RT, Beisel CL (2015) Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression. Nucleic Acids Res 43:674–681. https://doi.org/10.1093/nar/gku971
Mack D (2011) Probiotics in Inflammatory Bowel Diseases and Associated Conditions. Nutrients 3(2):245–264. https://doi.org/10.3390/nu3020245
Mavrich TN, Casey E, Oliveira J et al (2018) Characterization and induction of prophages in human gut-associated Bifidobacterium hosts. Sci Rep 8:12772. https://doi.org/10.1038/s41598-018-31181-3
Mimee M, Citorik RJ, Lu TK (2016) Microbiome therapeutics—Advances and challenges. Adv Drug Deliv Rev 105:44–54. https://doi.org/10.1016/j.addr.2016.04.032
Miro-Blanch, J., & Yanes, O. (2019). Epigenetic regulation at the interplay between gut microbiota and host metabolism. Front Genet, 10. https://doi.org/10.3389/fgene.2019.00638
Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV, van der Oost J (2016) Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science 353(6299):aad5147. https://doi.org/10.1126/science.aad5147
Moradpour Z, Sepehrizadeh Z, Rahbarizadeh F, Ghasemian A, Yazdi MT, Shahverdi AR (2009) Genetically engineered phage harbouring the lethal catabolite gene activator protein gene with an inducer-independent promoter for biocontrol of Escherichia coli. FEMS Microbiol Lett 296:67–71. https://doi.org/10.1111/j.1574-6968.2009.01620.x
Myrbråten, I. S., Wiull, K., Salehian, Z., Håvarstein, L. S., Straume, D., Mathiesen, G., & Kjos, M. (2019). CRISPR interference for rapid knockdown of essential cell cycle genes in Lactobacillus plantarum. MSphere, 4(2), e00007-19, /msphere/4/2/mSphere007-19.atom. https://doi.org/10.1128/mSphere.00007-19
Nagpal R, Kumar A, Kumar M, Behare PV, Jain S, Yadav H (2012) Probiotics, their health benefits and applications for developing healthier foods: a review. FEMS Microbiol Lett 334(1):1–15. https://doi.org/10.1111/j.1574-6968.2012.02593.x
Oh J-H, van Pijkeren J-P (2014) CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res 42(17):e131–e131. https://doi.org/10.1093/nar/gku623
Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173–1183. https://doi.org/10.1016/j.cell.2013.02.022
Ramírez AM, Rodriguez-López A, Ardila A, Beltran L, Patarroyo CA, Melendez ADP, Sánchez OF, Alméciga-Díaz CJ (2017) Production of human recombinant phenylalanine hydroxylase in Lactobacillus plantarum for gastrointestinal delivery. Eur J Pharm Sci 109:48–55. https://doi.org/10.1016/j.ejps.2017.07.033
Rodrigues M, McBride SW, Hullahalli K, Palmer KL, Duerkop BA (2019) Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci [Preprint]. Microbiol. https://doi.org/10.1101/678573
Rosenbluh J, Xu H, Harrington W, Gill S, Wang X, Vazquez F, Root DE, Tsherniak A, Hahn WC (2017) Complementary information derived from CRISPR Cas9 mediated gene deletion and suppression. Nat Comm 8(1):15403. https://doi.org/10.1038/ncomms15403
Sakaguchi K, Funaoka N, Tani S et al (2013) The pyrE gene as a bidirectional selection marker in Bifidobacterium longum 105-A. Biosci Microbiota Food Health 32(2):59–68. https://doi.org/10.12938/bmfh.32.59
Sedighi M, Zahedi Bialvaei A, Hamblin MR, Ohadi E, Asadi A, Halajzadeh M, Lohrasbi V, Mohammadzadeh N, Amiriani T, Krutova M, Amini A, Kouhsari E (2019) Therapeutic bacteria to combat cancer; current advances, challenges, and opportunities. Cancer Med 8(6):3167–3181. https://doi.org/10.1002/cam4.2148
Sleytr UB, Schuster B, Egelseer EM, Pum D (2014) S-layers: principles and applications. FEMS Microbiol Rev 38:823–864. https://doi.org/10.1111/1574-6976.12063
Turer E, McAlpine W, Wang K, Lu T, Li X, Tang M, Zhan X, Wang T, Zhan X, Bu C-H, Murray AR, Beutler B (2017) Creatine maintains intestinal homeostasis and protects against colitis. PNAS 114(7):E1273–E1281. https://doi.org/10.1073/pnas.1621400114
Volzing K, Borrero J, Sadowsky MJ, Kaznessis YN (2013) Antimicrobial peptides targeting Gram-negative pathogens, produced and delivered by lactic acid bacteria. ACS synthc biol 2(11):643–650. https://doi.org/10.1021/sb4000367
Wu Q, Shah NP (2017) The potential of species-specific tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group for galactose reduction in fermented dairy foods. Food Microbiol 62:178–187. https://doi.org/10.1016/j.fm.2016.10.027
Xiong Z-Q, Wei Y-Y, Kong L-H, Song X, Yi H-X, Ai L-Z (2020) Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis. J Dairy Sci 103(1):161–165. https://doi.org/10.3168/jds.2019-17346
Yadav R, Kumar V, Baweja M, Shukla P (2018) Gene editing and genetic engineering approaches for advanced probiotics: A review. Crit Rev Food Sci Nutr 58(10):1735–1746. https://doi.org/10.1080/10408398.2016.1274877
Yosef I, Manor M, Kiro R, Qimron U (2015) Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. PNAS 112(23):7267–7272. https://doi.org/10.1073/pnas.1500107112
Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La Russa M, Tsai JC, Weissman JS, Dueber JE, Qi LS, Lim WA (2015) Engineering complex synthetic transcriptional programs with crispr rna scaffolds. Cell 160(1–2):339–350. https://doi.org/10.1016/j.cell.2014.11.052
Zhou D, Jiang Z, Pang Q, Zhu Y, Wang Q, Qi Q (2019) CRISPR/Cas9-assisted seamless genome editing in Lactobacillus plantarum and its application in N -acetylglucosamine production. Appl Environ Microbiol 85(21):e01367-e1419. https://doi.org/10.1128/AEM.01367-19
Zhou Z, Chen X, Sheng H, Shen X, Sun X, Yan Y, Wang J, Yuan Q (2020) Engineering probiotics as living diagnostics and therapeutics for improving human health. Microb Cell Fact 19(1):56. https://doi.org/10.1186/s12934-020-01318-z
Zuo F, Marcotte H (2021) Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing. Curr Opin Biotechnol 70:75–82. https://doi.org/10.1016/j.copbio.2020.12.015
Zuo, F., Zeng, Z., Hammarström, L., & Marcotte, H. (2019). Inducible plasmid self-destruction (IPSD) assisted genome engineering in lactobacilli and bifidobacteria. ACS synth biol, 8(8), 1723-1729. https://doi/full/https://doi.org/10.1021/acssynbio.9b00114
Acknowledgements
Authors thank The Director, CSIR- Central Food Technological Research Institute, Mysuru for facilities and encouragement to write the article. AS would like to thank CSIR for providing Nehru Post- Doctoral Fellowship. We acknowledge Drs. MS Gopinath and SVN Vijayendra for their critical comments on the manuscript.
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Sundararaman, A., Halami, P.M. Genome editing of probiotic bacteria: present status and future prospects. Biologia 77, 1831–1841 (2022). https://doi.org/10.1007/s11756-022-01049-z
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DOI: https://doi.org/10.1007/s11756-022-01049-z