A single plasmid based CRISPR interference in Synechocystis 6803 – A proof of concept

Autoři: Prithwiraj Kirtania aff001;  Barbara Hódi aff001;  Ivy Mallick aff001;  István Zoltan Vass aff001;  Tamás Fehér aff003;  Imre Vass aff001;  Peter B. Kós aff001
Působiště autorů: Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary aff001;  Doctoral School of Biology, University of Szeged, Szeged, Hungary aff002;  Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary aff003;  Department of Biotechnology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225375


We developed a simple method to apply CRISPR interference by modifying an existing plasmid pCRISPathBrick containing the native S. pyogenes CRISPR assembly for Synechocystis PCC6803 and named it pCRPB1010. The technique presented here using deadCas9 is easier to implement for gene silencing in Synechocystis PCC6803 than other existing techniques as it circumvents the genome integration and segregation steps thereby significantly shortens the construction of the mutant strains. We executed CRISPR interference against well characterized photosynthetic genes to get a clear phenotype to validate the potential of pCRPB1010 and presented the work as a “proof of concept”. Targeting the non-template strand of psbO gene resulted in decreased amount of PsbO and 50% decrease in oxygen evolution rate. Targeting the template strand of psbA2 and psbA3 genes encoding the D1 subunit of photosystem II (PSII) using a single spacer against the common sequence span of the two genes, resulted in full inhibition of both genes, complete abolition of D1 protein synthesis, complete loss of oxygen evolution as well as photoautotrophic growth arrest. This is the first report of a single plasmid based, completely lesion free and episomal expression and execution of CRISPR interference in Synechocystis PCC6803.

Klíčová slova:

Antibiotics – CRISPR – Genetic interference – Oxygen – Plasmid construction – Polymerase chain reaction – Sequence motif analysis – Synechocystis


1. Bikard D, Marraffini LA. Control of gene expression by CRISPR-Cas systems. F1000Prime Rep. 2013;5:47. Epub 2013/11/26. doi: 10.12703/P5-47 24273648; PubMed Central PMCID: PMC3816762.

2. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–21. Epub 2012/06/30. doi: 10.1126/science.1225829 22745249; PubMed Central PMCID: PMC6286148.

3. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013;152(5):1173–83. Epub 2013/03/05. doi: 10.1016/j.cell.2013.02.022 23452860; PubMed Central PMCID: PMC3664290.

4. Didovyk A, Borek B, Tsimring L, Hasty J. Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications. Current opinion in biotechnology. 2016;40:177–84. Epub 2016/06/23. doi: 10.1016/j.copbio.2016.06.003 27344519.

5. Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41(15):7429–37. Epub 2013/06/14. doi: 10.1093/nar/gkt520 23761437; PubMed Central PMCID: PMC3753641.

6. Xu X, Qi LS. A CRISPR-dCas Toolbox for Genetic Engineering and Synthetic Biology. J Mol Biol. 2019;431(1):34–47. doi: 10.1016/j.jmb.2018.06.037 29958882.

7. van der Oost J, Swarts DC, Jore MM. Prokaryotic Argonautes—variations on the RNA interference theme. Microb Cell. 2014;1(5):158–9. Epub 2014/04/15. doi: 10.15698/mic2014.05.144 28357239; PubMed Central PMCID: PMC5354601.

8. Yao L, Cengic I, Anfelt J, Hudson EP. Multiple Gene Repression in Cyanobacteria Using CRISPRi. ACS Synth Biol. 2016;5(3):207–12. Epub 2015/12/23. doi: 10.1021/acssynbio.5b00264 26689101.

9. Cress BF, Toparlak OD, Guleria S, Lebovich M, Stieglitz JT, Englaender JA, et al. CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli. ACS Synth Biol. 2015;4(9):987–1000. Epub 2015/03/31. doi: 10.1021/acssynbio.5b00012 25822415.

10. Nyerges A, Balint B, Cseklye J, Nagy I, Pal C, Feher T. CRISPR-interference based modulation of mobile genetic elements in bacteria. Synthetic Biology. 2019;4:In Press. doi: 10.1101/428029%J bioRxiv

11. Al-Haj L, Lui YT, Abed RMM, Gomaa MA, Purton S. Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects. Life (Basel, Switzerland). 2016;6(4):42. doi: 10.3390/life6040042 27916886.

12. Nogales J, Gudmundsson S, Knight EM, Palsson BO, Thiele I. Detailing the optimality of photosynthesis in cyanobacteria through systems biology analysis. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(7):2678–83. doi: 10.1073/pnas.1117907109 %J Proceedings of the National Academy of Sciences. 22308420

13. Zerulla K, Ludt K, Soppa J. The ploidy level of Synechocystis sp. PCC 6803 is highly variable and is influenced by growth phase and by chemical and physical external parameters. Microbiology. 2016;162(5):730–9. Epub 2016/02/28. doi: 10.1099/mic.0.000264 26919857.

14. Li H, Shen CR, Huang CH, Sung LY, Wu MY, Hu YC. CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production. Metab Eng. 2016;38:293–302. Epub 2016/10/30. doi: 10.1016/j.ymben.2016.09.006 27693320.

15. Behler J, Vijay D, Hess WR, Akhtar MK. CRISPR-Based Technologies for Metabolic Engineering in Cyanobacteria. Trends Biotechnol. 2018;36(10):996–1010. Epub 2018/06/26. doi: 10.1016/j.tibtech.2018.05.011 29937051.

16. Naduthodi MIS, Barbosa MJ, van der Oost J. Progress of CRISPR-Cas Based Genome Editing in Photosynthetic Microbes. Biotechnology Journal. 2018;13(9):1700591. doi: 10.1002/biot.201700591 29396999

17. Ungerer J, Pakrasi HB. Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria. Sci Rep. 2016;6:39681. doi: 10.1038/srep39681 28000776; PubMed Central PMCID: PMC5175191.

18. Wendt KE, Ungerer J, Cobb RE, Zhao H, Pakrasi HB. CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. Microb Cell Fact. 2016;15(1):115. Epub 2016/06/25. doi: 10.1186/s12934-016-0514-7 27339038; PubMed Central PMCID: PMC4917971.

19. Shabestary K, Anfelt J, Ljungqvist E, Jahn M, Yao L, Hudson EP. Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria. ACS Synth Biol. 2018;7(7):1669–75. Epub 2018/06/08. doi: 10.1021/acssynbio.8b00056 29874914.

20. Kaczmarzyk D, Cengic I, Yao L, Hudson EP. Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX. Metab Eng. 2018;45:59–66. Epub 2017/12/05. doi: 10.1016/j.ymben.2017.11.014 29199103.

21. Labarre J, Chauvat F, Thuriaux P. Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803. J Bacteriol. 1989;171(6):3449–57. Epub 1989/06/01. doi: 10.1128/jb.171.6.3449-3457.1989 2498291; PubMed Central PMCID: PMC210070.

22. Vass I, Cook KM, Deák Z, mayes SR, Barber J. Thermoluminescence and flash-oxygen characterization of the IC2 deletion mutant of Synechocystis sp. PCC 6803 lacking the Photosystem II 33 kDa protein. Biochimica et Biophysica Acta (BBA)—Bioenergetics. 1992;1102(2):195–201. https://doi.org/10.1016/0005-2728(92)90100-G.

23. Komenda J, Reisinger V, Muller BC, Dobakova M, Granvogl B, Eichacker LA. Accumulation of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center complex in Synechocystis PCC 6803. The Journal of biological chemistry. 2004;279(47):48620–9. Epub 2004/09/07. doi: 10.1074/jbc.M405725200 15347679.

24. Higo A, Isu A, Fukaya Y, Ehira S, Hisabori T. Application of CRISPR Interference for Metabolic Engineering of the Heterocyst-Forming Multicellular Cyanobacterium Anabaena sp. PCC 7120. Plant Cell Physiol. 2018;59(1):119–27. doi: 10.1093/pcp/pcx166 29112727.

25. Komenda J, Barber J. Comparison of psbO and psbH deletion mutants of Synechocystis PCC 6803 indicates that degradation of D1 protein is regulated by the Q(B)site and dependent on protein-synthesis. Biochemistry. 1995;34(29):9625–31. doi: 10.1021/bi00029a040 WOS:A1995RL61000040. 7626631

26. Liang F, Lindblad P. Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803. Metab Eng. 2016;38:56–64. doi: 10.1016/j.ymben.2016.06.005 27328433.

27. Powell B, Mergeay M, Christofi N. Transfer of broad host-range plasmids to sulphate-reducing bacteria. FEMS Microbiology Letters. 1989;59(3):269–73. doi: 10.1111/j.1574-6968.1989.tb03123.x

28. Guerrero F, Carbonell V, Cossu M, Correddu D, Jones PR. Ethylene synthesis and regulated expression of recombinant protein in Synechocystis sp. PCC 6803. PLoS One. 2012;7(11):e50470. Epub 2012/11/28. doi: 10.1371/journal.pone.0050470 23185630; PubMed Central PMCID: PMC3503970.

29. Clerico EM, Ditty JL, Golden SS. Specialized techniques for site-directed mutagenesis in cyanobacteria. Methods Mol Biol. 2007;362:155–71. Epub 2007/04/10. doi: 10.1007/978-1-59745-257-1_11 17417008.

30. Barbato R, Friso G, Giardi MT, Rigoni F, Giacometti GM. Breakdown of the photosystem II reaction center D1 protein under photoinhibitory conditions: identification and localization of the C-terminal degradation products. Biochemistry. 1991;30(42):10220–6. Epub 1991/10/22. doi: 10.1021/bi00106a021 1931951.

31. Mougiakos I, Bosma EF, Ganguly J, van der Oost J, van Kranenburg R. Hijacking CRISPR-Cas for high-throughput bacterial metabolic engineering: advances and prospects. Curr Opin Biotechnol. 2018;50:146–57. doi: 10.1016/j.copbio.2018.01.002 29414054.

32. Elhai J, Wolk CP. A Versatile Class of Positive-Selection Vectors Based on the Nonviability of Palindrome-Containing Plasmids That Allows Cloning into Long Polylinkers. Gene. 1988;68(1):119–38. doi: 10.1016/0378-1119(88)90605-1 WOS:A1988P913000013. 2851487

33. Sicora CI, Ho FM, Salminen T, Styring S, Aro EM. Transcription of a "silent" cyanobacterial psbA gene is induced by microaerobic conditions. Biochim Biophys Acta. 2009;1787(2):105–12. doi: 10.1016/j.bbabio.2008.12.002 19124001.

34. Mate Z, Sass L, Szekeres M, Vass I, Nagy F. UV-B-induced differential transcription of psbA genes encoding the D1 protein of photosystem II in the Cyanobacterium synechocystis 6803. The Journal of biological chemistry. 1998;273(28):17439–44. doi: 10.1074/jbc.273.28.17439 9651331.

35. Cleto S, Jensen JVK, Wendisch VF, Lu TK. Corynebacterium glutamicum Metabolic Engineering with CRISPR Interference (CRISPRi). Acs Synthetic Biology. 2016;5(5):375–85. doi: 10.1021/acssynbio.5b00216 WOS:000376476900002. 26829286

36. Jansson C, Debus RJ, Osiewacz HD, Gurevitz M, McIntosh L. Construction of an Obligate Photoheterotrophic Mutant of the Cyanobacterium Synechocystis 6803: Inactivation of the psbA Gene Family. Plant Physiol. 1987;85(4):1021–5. Epub 1987/12/01. doi: 10.1104/pp.85.4.1021 16665796; PubMed Central PMCID: PMC1054386.

37. Nixon PJ, Rogner M, Diner BA. Expression of a higher plant psbA gene in Synechocystis 6803 yields a functional hybrid photosystem II reaction center complex. Plant Cell. 1991;3(4):383–95. Epub 1991/04/01. doi: 10.1105/tpc.3.4.383 1840918; PubMed Central PMCID: PMC160008.

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