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Microbial Genomics logo Microbial Genomics

Volume 8, Issue 4

Characterization of CRISPR-Cas systems in Open Access

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system is an important adaptive immune system for bacteria to resist foreign DNA infection, which has been widely used in genotyping and gene editing. To provide a theoretical basis for the application of the CRISPR-Cas system in , the occurrence and diversity of CRISPR-Cas systems were analysed in 150 strains. Specifically, 47 % (71/150) of genomes possessed the CRISPR-Cas system, and type I-C CRISPR-Cas system was the most widely distributed among those strains. The spacer sequences present in can be used as a genotyping marker. Additionally, the phage assembly-related proteins were important targets of the type I-C CRISPR-Cas system in , and the protospacer adjacent motif sequences were further characterized in type I-C system as 5′-TTC-3′. All these results might provide a molecular basis for the development of endogenous genome editing tools in .

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Bifidobacterium breve , CRISPR-Cas system , genotyping and prophage
Funding
This study was supported by the:
  • Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province (Award Not Applicable)
    • Principle Award Recipient: JianxinZhao
  • National Natural Science Foundation of China (Award 31820103010)
    • Principle Award Recipient: WeiChen
  • 111 Project (Award BP0719028)
    • Principle Award Recipient: BoYang
  • National Natural Science Foundation of China (Award Nos. 32021005)
    • Principle Award Recipient: BoYang
© 2022 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License.
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2022-04-22
2025-04-26
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References

  1. Kelly D, King T, Aminov R. Importance of microbial colonization of the gut in early life to the development of immunity. Mutat Res 2007; 622:58–69 [View Article] [PubMed]
     [Google Scholar]
  2. Lloyd-Price J, Abu-Ali G, Huttenhower C. The healthy human microbiome. Genome Med 2016; 8:1–11 [View Article] [PubMed]
     [Google Scholar]
  3. Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 2020; 18:67–83 [View Article] [PubMed]
     [Google Scholar]
  4. Goh YJ, Barrangou R. Harnessing CRISPR-Cas systems for precision engineering of designer probiotic lactobacilli. Curr Opin Biotechnol 2019; 56:163–171 [View Article] [PubMed]
     [Google Scholar]
  5. Jiang W, Zhou H, Bi H, Fromm M, Yang B et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 2013; 41:20 [View Article] [PubMed]
     [Google Scholar]
  6. Harms DW, Quadros RM, Seruggia D, Ohtsuka M, Takahashi G et al. Mouse genome editing using the CRISPR/Cas system. Curr Protoc Hum Genet 2014; 83:15 [View Article] [PubMed]
     [Google Scholar]
  7. Giacalone JC, Sharma TP, Burnight ER, Fingert JF, Mullins RF et al. CRISPR-Cas9-based genome editing of human induced pluripotent stem cells. Curr Protoc Stem Cell Biol 2018; 44:5B [View Article] [PubMed]
     [Google Scholar]
  8. Hidalgo-Cantabrana C, Delgado S, Ruiz L, Ruas-Madiedo P, Sánchez B et al. Bifidobacteria and their health-promoting effects. Microbiol Spectr 2017; 5: [View Article] [PubMed]
     [Google Scholar]
  9. Turroni F, Peano C, Pass DA, Foroni E, Severgnini M et al. Diversity of bifidobacteria within the infant gut microbiota. PLoS One 2012; 7:e36957 [View Article] [PubMed]
     [Google Scholar]
  10. Braga TD, da Silva GAP, de Lira PIC, de Carvalho Lima M. Efficacy of Bifidobacterium breve and Lactobacillus casei oral supplementation on necrotizing enterocolitis in very-low-birth-weight preterm infants: a double-blind, randomized, controlled trial. Am J Clin Nutr 2011; 93:81–86 [View Article] [PubMed]
     [Google Scholar]
  11. Klemenak M, Dolinšek J, Langerholc T, Di Gioia D, Mičetić-Turk D. Administration of Bifidobacterium breve decreases the production of TNF-α in children with celiac disease. Dig Dis Sci 2015; 60:3386–3392 [View Article]
     [Google Scholar]
  12. Solito A, Bozzi Cionci N, Calgaro M, Caputo M, Vannini L et al. Supplementation with Bifidobacterium breve BR03 and B632 strains improved insulin sensitivity in children and adolescents with obesity in a cross-over, randomized double-blind placebo-controlled trial. Clin Nutr 2021; 40:4585–4594 [View Article]
     [Google Scholar]
  13. Enomoto T, Sowa M, Nishimori K, Shimazu S, Yoshida A et al. Effects of bifidobacterial supplementation to pregnant women and infants in the prevention of allergy development in infants and on fecal microbiota. Allergol Int 2014; 63:575–585 [View Article]
     [Google Scholar]
  14. Misra CS, Bindal G, Sodani M, Wadhawan S, Kulkarni S et al. Determination of Cas9/dCas9 associated toxicity in microbes. Microbiology 2019848135 [View Article]
     [Google Scholar]
  15. Wurihan W, Huang Y, Weber AM, Wu X, Fan H. Nonspecific toxicities of Streptococcus pyogenes and Staphylococcus aureus dCas9 in Chlamydia trachomatis. Pathog Dis 2019; 77:ftaa005 [View Article]
     [Google Scholar]
  16. Cho S, Choe D, Lee E, Kim SC, Palsson B et al. High-level dCas9 expression induces abnormal cell morphology in Escherichia coli. ACS Synth Biol 2018; 7:1085–1094 [View Article] [PubMed]
     [Google Scholar]
  17. Bottacini F, Morrissey R, Roberts RJ, James K, van Breen J et al. Comparative genome and methylome analysis reveals restriction/modification system diversity in the gut commensal Bifidobacterium breve. Nucleic Acids Res 2018; 46:1860–1877 [View Article] [PubMed]
     [Google Scholar]
  18. O’ Connell Motherway M, Watson D, Bottacini F, Clark TA, Roberts RJ et al. Identification of restriction-modification systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT sequencing and associated methylome analysis. PLoS One 2014; 9:e94875 [View Article] [PubMed]
     [Google Scholar]
  19. Liu R, Yang B, Stanton C, Paul Ross R, Zhao J et al. Comparative genomics and gene-trait matching analysis of Bifidobacterium breve from Chinese children. Food Biosci 2020; 36:100631 [View Article]
     [Google Scholar]
  20. Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J et al. CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res 2018; 46:W246–W251 [View Article] [PubMed]
     [Google Scholar]
  21. Biswas A, Staals RHJ, Morales SE, Fineran PC, Brown CM. CRISPRDetect: A flexible algorithm to define CRISPR arrays. BMC Genomics 2016; 17:356 [View Article] [PubMed]
     [Google Scholar]
  22. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 2018; 35:1547–1549 [View Article] [PubMed]
     [Google Scholar]
  23. Hidalgo-Cantabrana C, Crawley AB, Sanchez B, Barrangou R. Characterization and exploitation of CRISPR Loci in Bifidobacterium longum. Front Microbiol 2017; 8:1851 [View Article] [PubMed]
     [Google Scholar]
  24. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 2021; 49:W293–W296 [View Article] [PubMed]
     [Google Scholar]
  25. Arndt D, Grant JR, Marcu A, Sajed T, Pon A et al. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res 2016; 44:W16–21 [View Article] [PubMed]
     [Google Scholar]
  26. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant 2020; 13:1194–1202 [View Article] [PubMed]
     [Google Scholar]
  27. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014; 30:2068–2069 [View Article] [PubMed]
     [Google Scholar]
  28. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res 2004; 14:1188–1190 [View Article] [PubMed]
     [Google Scholar]
  29. Briner AE, Lugli GA, Milani C, Duranti S, Turroni F et al. Occurrence and diversity of CRISPR-cas systems in the genus Bifidobacterium. PLoS One 2015; 10:e0133661 [View Article] [PubMed]
     [Google Scholar]
  30. Crawley AB, Henriksen ED, Stout E, Brandt K, Barrangou R. Characterizing the activity of abundant, diverse and active CRISPR-Cas systems in lactobacilli. Sci Rep 2018; 8:11544 [View Article] [PubMed]
     [Google Scholar]
  31. Makarova KS, Haft DH, Barrangou R, Brouns SJJ, Charpentier E et al. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 2011; 9:467–477 [View Article] [PubMed]
     [Google Scholar]
  32. Leon LM, Mendoza SD, Bondy-Denomy J. How bacteria control the CRISPR-Cas arsenal. Curr Opin Microbiol 2018; 42:87–95 [View Article] [PubMed]
     [Google Scholar]
  33. Garrett SC. Pruning and tending immune memories: spacer dynamics in the CRISPR array. Front Microbiol 2021; 12:664299 [View Article] [PubMed]
     [Google Scholar]
  34. Shah SA, Erdmann S, Mojica FJM, Garrett RA. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol 2013; 10:891–899 [View Article] [PubMed]
     [Google Scholar]
  35. Bozzi Cionci N, Baffoni L, Gaggìa F, Di Gioia D. Therapeutic microbiology: the role of Bifidobacterium breve as food supplement for the prevention/treatment of paediatric diseases. Nutrients 2018; 10:E1723 [View Article] [PubMed]
     [Google Scholar]
  36. Manrique P, Dills M, Young MJ. The human gut phage community and its implications for health and disease. Viruses 2017; 9:E141 [View Article]
     [Google Scholar]
  37. Crawley AB, Henriksen JR, Barrangou R. CRISPRdisco: an automated pipeline for the discovery and analysis of CRISPR-Cas systems. CRISPR J 2018; 1:171–181 [View Article]
     [Google Scholar]
  38. Pan M, Nethery MA, Hidalgo-Cantabrana C, Barrangou R. Comprehensive mining and characterization of CRISPR-cas systems in Bifidobacterium. Microorganisms 2020; 8:720 [View Article]
     [Google Scholar]
  39. Wang G, Liu Q, Pei Z, Wang L, Tian P et al. The diversity of the CRISPR-Cas system and prophages present in the genome reveals the co-evolution of Bifidobacterium pseudocatenulatum and phages. Front Microbiol 2020; 11:1088 [View Article]
     [Google Scholar]
  40. Li M, Gong L, Cheng F, Yu H, Zhao D et al. Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems. Science 2021; 372:eabe5601 [View Article]
     [Google Scholar]
  41. Wang J, Li J, Zhao H, Sheng G, Wang M et al. Structural and mechanistic basis of PAM-dependent spacer acquisition in CRISPR-Cas systems. Cell 2015; 163:840–853 [View Article] [PubMed]
     [Google Scholar]
  42. Rollie C, Schneider S, Brinkmann AS, Bolt EL, White MF. Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition. Elife 2015; 4: [View Article] [PubMed]
     [Google Scholar]
  43. Nuñez JK, Lee ASY, Engelman A, Doudna JA. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity. Nature 2015; 519:193–198 [View Article] [PubMed]
     [Google Scholar]
  44. Huo Y, Nam KH, Ding F, Lee H, Wu L et al. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation. Nat Struct Mol Biol 2014; 21:771–777 [View Article] [PubMed]
     [Google Scholar]
  45. Wang L, Wang Y, Li Q, Tian K, Xu L et al. Exopolysaccharide, isolated from a novel strain Bifidobacterium breve lw01 possess an anticancer effect on head and neck cancer - genetic and biochemical evidences. Front Microbiol 2019; 10:1044 [View Article] [PubMed]
     [Google Scholar]
  46. Zhou X, Yang B, Stanton C, Ross RP, Zhao J et al. Comparative analysis of Lactobacillus gasseri from Chinese subjects reveals a new species-level taxa. BMC Genomics 2020; 21:119 [View Article] [PubMed]
     [Google Scholar]
  47. Hille F, Charpentier E. CRISPR-Cas: biology, mechanisms and relevance. Philos Trans R Soc Lond B Biol Sci 2016; 371:20150496 [View Article] [PubMed]
     [Google Scholar]
  48. Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 2008; 321:960–964 [View Article] [PubMed]
     [Google Scholar]
  49. Sorek R, Kunin V, Hugenholtz P. CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol 2008; 6:181–186 [View Article] [PubMed]
     [Google Scholar]
  50. Kunin V, Sorek R, Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol 2007; 8:R61 [View Article] [PubMed]
     [Google Scholar]
  51. Marraffini LA, Sontheimer EJ. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 2010; 463:568–571 [View Article] [PubMed]
     [Google Scholar]
  52. Mohanraju P, Makarova KS, Zetsche B, Zhang F, Koonin EV et al. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science 2016; 353:aad5147 [View Article] [PubMed]
     [Google Scholar]
  53. Briner AE, Barrangou R. Lactobacillus buchneri genotyping on the basis of clustered regularly interspaced short palindromic repeat (CRISPR) locus diversity. Appl Environ Microbiol 2014; 80:994–1001 [View Article] [PubMed]
     [Google Scholar]
  54. Zeng H, Li C, He W, Zhang J, Chen M et al. Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter dublinensis genotyping based on CRISPR locus diversity. Front Microbiol 2019; 10:1989 [View Article]
     [Google Scholar]
  55. Pei Z, Sadiq FA, Han X, Zhao J, Zhang H et al. Identification, characterization, and phylogenetic analysis of eight new inducible prophages in Lactobacillus. Virus Res 2020; 286:198003 [View Article] [PubMed]
     [Google Scholar]
  56. Lugli GA, Milani C, Turroni F, Tremblay D, Ferrario C et al. Prophages of the genus Bifidobacterium as modulating agents of the infant gut microbiota. Environ Microbiol 2016; 18:2196–2213 [View Article] [PubMed]
     [Google Scholar]
  57. Stern A, Keren L, Wurtzel O, Amitai G, Sorek R. Self-targeting by CRISPR: gene regulation or autoimmunity?. Trends Genet 2010; 26:335–340 [View Article] [PubMed]
     [Google Scholar]
  58. Levy A, Goren MG, Yosef I, Auster O, Manor M et al. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 2015; 520:505–510 [View Article] [PubMed]
     [Google Scholar]
  59. Stanley SY, Borges AL, Chen K-H, Swaney DL, Krogan NJ et al. Anti-CRISPR-associated proteins are crucial repressors of anti-CRISPR transcription. Cell 2019; 178:1452–1464 [View Article] [PubMed]
     [Google Scholar]
  60. Katsura I, Hendrix RW. Length determination in bacteriophage lambda tails. Cell 1984; 39:691–698 [View Article] [PubMed]
     [Google Scholar]
  61. Murphy KC. Phage recombinases and their applications. Adv Virus Res 2012; 83:367–414 [View Article] [PubMed]
     [Google Scholar]
  62. Oh B, Moyer CL, Hendrix RW, Duda RL. The delta domain of the HK97 major capsid protein is essential for assembly. Virology 2014; 456–457:171–178 [View Article] [PubMed]
     [Google Scholar]
  63. Kala S, Cumby N, Sadowski PD, Hyder BZ, Kanelis V et al. HNH proteins are a widespread component of phage DNA packaging machines. Proc Natl Acad Sci U S A 2014; 111:6022–6027 [View Article] [PubMed]
     [Google Scholar]
  64. Aksyuk AA, Rossmann MG. Bacteriophage assembly. Viruses 2011; 3:172–203 [View Article] [PubMed]
     [Google Scholar]
  65. Oh J-H, van Pijkeren J-P. CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res 2014; 42:e131 [View Article] [PubMed]
     [Google Scholar]
  66. Song X, Huang H, Xiong Z, Ai L, Yang S. CRISPR-Cas9D10A nickase-assisted genome editing in Lactobacillus casei. Appl Environ Microbiol 2017; 83:e01259-17 [View Article] [PubMed]
     [Google Scholar]
  67. de Maat V, Stege PB, Dedden M, Hamer M, van Pijkeren J-P et al. CRISPR-Cas9-mediated genome editing in vancomycin-resistant Enterococcus faecium. FEMS Microbiol Lett 2019; 366:fnz256 [View Article] [PubMed]
     [Google Scholar]
  68. Guo T, Xin Y, Zhang Y, Gu X, Kong J. A rapid and versatile tool for genomic engineering in Lactococcus lactis. Microb Cell Fact 2019; 18:22 [View Article] [PubMed]
     [Google Scholar]
  69. O’Connell Motherway M, O’Driscoll J, Fitzgerald GF, Van Sinderen D. Overcoming the restriction barrier to plasmid transformation and targeted mutagenesis in Bifidobacterium breve UCC2003. Microb Biotechnol 2009; 2:321–332 [View Article] [PubMed]
     [Google Scholar]
  70. Hidalgo-Cantabrana C, Goh YJ, Pan M, Sanozky-Dawes R, Barrangou R. Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus. Proc Natl Acad Sci U S A 2019; 116:15774–15783 [View Article] [PubMed]
     [Google Scholar]
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Characterization of CRISPR-Cas systems in Bifidobacterium breve
M Gen 8, 000812 (2022); https://doi.org/10.1099/mgen.0.000812
/content/journal/mgen/10.1099/mgen.0.000812
/content/journal/mgen/10.1099/mgen.0.000812
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