Bioinformatics investigation of CRISPR/Cas systems in Bifidobacterium longum

Document Type : Research(Original) Article

Authors

1 Pharmaceutical Sciences Research Center, Shiraz University of Medical Science, Shiraz, Iran

2 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.

Abstract

Bifidobacterium longum is one of the most important probiotics used in the food industries. These bacteria are also applied for the treatment or prevention of gastrointestinal and gynecological diseases as well the regulation of immune system. On the other hand, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) systems has revolutionized gene therapy approaches and provided a novel tool for gene editing. Several investigators are seeking for potential CRISPR sequences as well the CRISPR-associated (Cas) proteins in various microorganism. Considering the importance of B. longum and its safe application in food industry, the aim of the present study is to investigate the presence and characteristics of CRISPRs in this bacterium via in silico study in 96 strains of the bacteria. According to the results obtained in this study, nine B. longum strains demonstrated high occurrence of CRISPR arrays. These arrays could probably form four RNA secondary structures. Since the bacterium B. longum have been widely used as a safe microorganism in foods, the first report on the probable presence of CRISPR sequences may provide the opportunity to introduce the bacteria as a gene editing tool.

Keywords


1. Hidalgo-Cantabrana C, Delgado S, Ruiz L, Ruas-Madiedo P, Sánchez B, Margolles A. Bifidobacteria and Their Health-Promoting Effects. Microbiol Spectr. 2017 Jun;5(3). doi: 10.1128/microbiolspec.BAD-0010-2016. PMID: 28643627.
2. Ventura M, Turroni F, Lugli GA, van Sinderen D. Bifidobacteria and humans: our special friends, from ecological to genomics perspectives. J Sci Food Agric. 2014 Jan 30;94(2):163-8. doi: 10.1002/jsfa.6356. Epub 2013 Sep 16. PMID: 23963950.
3. Freitas AC, Hill JE. Bifidobacteria isolated from vaginal and gut microbiomes are indistinguishable by comparative genomics. PLoS One. 2018 Apr 23;13(4):e0196290. doi: 10.1371/journal.pone.0196290. PMID: 29684056; PMCID: PMC5912743.
4. Zhu G , Ma F , Wang G , Wang Y , Zhao J , Zhang H , Chen W . Bifidobacteria attenuate the development of metabolic disorders, with inter- and intra-species differences. Food Funct. 2018 Jun 20;9(6):3509-3522. doi: 10.1039/c8fo00100f. PMID: 29892745.
5. Landete JM, Arqués JL. Fluorescent Lactic Acid Bacteria and Bifidobacteria as Vehicles of DNA Microbial Biosensors. Int J Mol Sci. 2017;18(8):1728. Published 2017 Aug 8. doi:10.3390/ijms18081728
6. Hidalgo-Cantabrana C, O'Flaherty S, Barrangou R. CRISPR-based engineering of next-generation lactic acid bacteria. Curr Opin Microbiol. 2017 Jun;37:79-87. doi: 10.1016/j.mib.2017.05.015. Epub 2017 Jun 13. PMID: 28622636.
7. Shi P, Qu H, Nian D, Chen Y, Liu X, Li Q, et al. Treatment of Guillain-Barré syndrome with Bifidobacterium infantis through regulation of T helper cells subsets. Int Immunopharmacol. 2018 Aug;61:290-296. doi: 10.1016/j.intimp.2018.06.015. Epub 2018 Jun 14. PMID: 29908492.
8. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014 Jun 5;157(6):1262-1278. doi: 10.1016/j.cell.2014.05.010. PMID: 24906146; PMCID: PMC4343198.
9. Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 2011 Jun;9(6):467-77. doi: 10.1038/nrmicro2577. Epub 2011 May 9. PMID: 21552286; PMCID: PMC3380444.
10. Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013;23(10):1163-71.
11. Wright AV, Nuñez JK, Doudna JA. Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering. Cell. 2016 Jan 14;164(1-2):29-44. doi: 10.1016/j.cell.2015.12.035. PMID: 26771484.
12. Makarova KS, Koonin EV. Annotation and Classification of CRISPR-Cas Systems. Methods Mol Biol. 2015;1311:47-75. doi: 10.1007/978-1-4939-2687-9_4. PMID: 25981466; PMCID: PMC5901762.
13. Stout E, Klaenhammer T, Barrangou R. CRISPR-Cas Technologies and Applications in Food Bacteria. Annu Rev Food Sci Technol. 2017 Feb 28;8:413-437. doi: 10.1146/annurev-food-072816-024723. PMID: 28245154.
14. Abriouel H, Pérez Montoro B, Casado Muñoz MDC, Knapp CW, Gálvez A, Benomar N. In silico genomic insights into aspects of food safety and defense mechanisms of a potentially probiotic Lactobacillus pentosus MP-10 isolated from brines of naturally fermented Aloreña green table olives. PLoS One. 2017 Jun 26;12(6):e0176801. doi: 10.1371/journal.pone.0176801. PMID: 28651019; PMCID: PMC5484467.
15. Li P, Li X, Gu Q, Lou XY, Zhang XM, Song DF, et al. Comparative genomic analysis of Lactobacillus plantarum ZJ316 reveals its genetic adaptation and potential probiotic profiles. J Zhejiang Univ Sci B. 2016 Aug;17(8):569-79. doi: 10.1631/jzus.B1600176. PMID: 27487802; PMCID: PMC4980435.
16. Selle K, Barrangou R. CRISPR-Based Technologies and the Future of Food Science. J Food Sci. 2015 Nov;80(11):R2367-72. doi: 10.1111/1750-3841.13094. Epub 2015 Oct 7. PMID: 26444151.
17. Briner AE, Lugli GA, Milani C, et al. Occurrence and Diversity of CRISPR-Cas Systems in the Genus Bifidobacterium. PLoS One. 2015;10(7):e0133661. Published 2015 Jul 31. doi:10.1371/journal.pone.0133661
18. Negahdaripour M, Nezafat N, Hajighahramani N, Rahmatabadi SS, Ghasemi Y. Investigating CRISPR-Cas systems in Clostridium botulinum via bioinformatics tools. Infect Genet Evol. 2017 Oct;54:355-373. doi: 10.1016/j.meegid.2017.06.027. Epub 2017 Jul 4. PMID: 28684374.
19. Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J, Néron B, et al. CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res. 2018 Jul 2;46(W1):W246-W251. doi: 10.1093/nar/gky425. PMID: 29790974; PMCID: PMC6030898.
20. Notredame C, Higgins DG, Heringa J. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000 Sep 8;302(1):205-17. doi: 10.1006/jmbi.2000.4042. PMID: 10964570.
21. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009 May 1;25(9):1189-91. doi: 10.1093/bioinformatics/btp033. Epub 2009 Jan 16. PMID: 19151095; PMCID: PMC2672624.
22. Lange SJ, Alkhnbashi OS, Rose D, Will S, Backofen R. CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems. Nucleic Acids Res. 2013;41(17):8034-8044. doi:10.1093/nar/gkt606
23. Hofacker IL. Vienna RNA secondary structure server. Nucleic Acids Res. 2003 Jul 1;31(13):3429-31. doi: 10.1093/nar/gkg599. PMID: 12824340; PMCID: PMC169005.
24. Elmer GW. Probiotics: "living drugs". Am J Health Syst Pharm. 2001;58(12):1101-9.
25. Lee ES, Song EJ, Nam YD, Lee SY. Probiotics in human health and disease: from nutribiotics to pharmabiotics. J Microbiol. 2018 Nov;56(11):773-782. doi: 10.1007/s12275-018-8293-y. Epub 2018 Oct 24. PMID: 30353462.
26. Picard C, Fioramonti J, Francois A, Robinson T, Neant F, Matuchansky C. Review article: bifidobacteria as probiotic agents -- physiological effects and clinical benefits. Aliment Pharmacol Ther. 2005 Sep 15;22(6):495-512. doi: 10.1111/j.1365-2036.2005.02615.x. PMID: 16167966.
27. Ouwehand AC, Sherwin S, Sindelar C, Smith AB, Stahl B. Production of Probiotic Bifidobacteria. The Bifidobacteria and Related Organisms: Elsevier; 2018. p. 261-9.
28. Scott A. How CRISPR is transforming drug discovery. Nature. 2018 Mar 8;555(7695):S10-S11. doi: 10.1038/d41586-018-02477-1. PMID: 29517026.
29. Fellmann C, Gowen BG, Lin P-C, Doudna JA, Corn JE. Cornerstones of CRISPR–Cas in drug discovery and therapy. Nat Rev Drug Discov. 2016;16:89.
30. Hidalgo-Cantabrana C, Crawley AB, Sanchez B, Barrangou R. Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum. Front Microbiol. 2017;8:1851. Published 2017 Sep 26. doi:10.3389/fmicb.2017.01851
31. Barrangou R, Doudna JA. Applications of CRISPR technologies in research and beyond. Nat Biotechnol. 2016;34:933.