Skip to main content
SpringerLink
Log in

Reprogramming CRISPR-Mediated RNA Interference for Silencing of Essential Genes in Sulfolobales

  • Protocol
  • First Online:
Archaea

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2522))

Abstract

The manipulation of gene expression levels in vivo is often key to elucidating gene function and regulatory network interactions, especially when it comes to the investigation of essential genes that cannot be deleted from the model organism’s genome. Several techniques have been developed for prokaryotes that allow to interfere with transcription initiation of specific genes by blocking or modifying promoter regions. However, a tool functionally similar to RNAi used in eukaryotes to efficiently degrade mRNA posttranscriptionally did not exist until recently. Type III CRISPR-Cas systems use small RNAs (crRNAs) that guide effector complexes (encoded by cas genes) which act as site-specific RNA endonuclease and can thus be harnessed for targeted posttranscriptional gene silencing. Guide RNAs complementary to the desired target mRNA that, in addition, exhibit complementarity to repeat sequences found in the CRISPR arrays, effectively suppress unspecific DNA and RNA activities of the CRISPR-Cas complexes. Here we describe the use of endogenous type III CRISPR-Cas systems in two model organisms of Crenarchaeota, Saccharolobus solfataricus and Sulfolobus acidocaldarius.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mohr S, Bakal C, Perrimon N (2010) Genomic screening with RNAi: results and challenges. Annu Rev Biochem 79:37–64

    Article  CAS  Google Scholar 

  2. Makarova KS, Wolf YI, Iranzo J et al (2020) Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 18:67–83. https://doi.org/10.1038/s41579-019-0299-x

    Article  CAS  PubMed  Google Scholar 

  3. Zink IA, Wimmer E, Schleper C (2020) Heavily armed ancestors: CRISPR immunity and applications in archaea with a comparative analysis of CRISPR types in sulfolobales. Biomol Ther 10:1–41. https://doi.org/10.3390/biom10111523

    Article  CAS  Google Scholar 

  4. Hille F, Richter H, Wong SP et al (2018) The biology of CRISPR-Cas: backward and forward. Cell 172:1239–1259. https://doi.org/10.1016/j.cell.2017.11.032

    Article  CAS  PubMed  Google Scholar 

  5. Jackson SA, McKenzie RE, Fagerlund RD et al (2017) CRISPR-Cas: adapting to change. Science 356:eaal5056. https://doi.org/10.1126/science.aal5056

    Article  CAS  PubMed  Google Scholar 

  6. Zhang X, Wang J, Wang J et al (2017) Multiplex gene regulation by CRISPR-ddCpf1. Cell Discov 3:1–9. https://doi.org/10.1038/celldisc.2017.18

    Article  CAS  Google Scholar 

  7. Qi LS, Larson MH, Gilbert LA et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. https://doi.org/10.1016/j.cell.2013.02.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gilbert LA, Larson MH, Morsut L et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154:442. https://doi.org/10.1016/j.cell.2013.06.044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Choudhary E, Thakur P, Pareek M, Agarwal N (2015) Gene silencing by CRISPR interference in mycobacteria. Nat Commun 6:6267. https://doi.org/10.1038/ncomms7267

    Article  CAS  PubMed  Google Scholar 

  10. Wensing L, Sharma J, Uthayakumar D et al (2019) A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans. mSphere 4:1–15. https://doi.org/10.1128/msphere.00002-19

    Article  CAS  Google Scholar 

  11. Myrbråten IS, Wiull K, Salehian Z et al (2019) CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes in Lactobacillus plantarum. mSphere 4:1–14. https://doi.org/10.1128/msphere.00007-19

    Article  CAS  Google Scholar 

  12. Zheng Y, Shen W, Zhang J et al (2018) CRISPR interference-based specific and efficient gene inactivation in the brain. Nat Neurosci 21:894. https://doi.org/10.1038/s41593-018-0125-1

    Article  CAS  PubMed  Google Scholar 

  13. Piatek A, Ali Z, Baazim H et al (2015) RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol J 13:578–589. https://doi.org/10.1111/pbi.12284

    Article  CAS  PubMed  Google Scholar 

  14. Stachler AE, Marchfelder A (2016) Gene repression in haloarchaea using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas I-B system. J Biol Chem 291:15226–15242. https://doi.org/10.1074/jbc.M116.724062

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 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

    Article  CAS  PubMed  Google Scholar 

  16. Elmore JR, Sheppard NF, Ramia N et al (2016) Bipartite recognition of target RNAs activates DNA cleavage by the type III-B CRISPR–Cas system. Genes Dev 30:447–459. https://doi.org/10.1101/gad.272153.115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kazlauskiene M, Tamulaitis G, Kostiuk G et al (2016) Spatiotemporal control of type III-A CRISPR-Cas immunity: coupling DNA degradation with the target RNA recognition. Mol Cell 62:295–306. https://doi.org/10.1016/j.molcel.2016.03.024

    Article  CAS  PubMed  Google Scholar 

  18. Han W, Li Y, Deng L et al (2016) A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction. Nucleic Acids Res:gkw1274. https://doi.org/10.1093/nar/gkw1274

  19. Samai P, Pyenson N, Jiang W et al (2015) Co-transcriptional DNA and RNA cleavage during type III CRISPR-Cas immunity. Cell 161:1164–1174. https://doi.org/10.1016/j.cell.2015.04.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Estrella MA, Kuo F, Bailey S (2016) RNA-activated DNA cleavage by the type III-B CRISPR—Cas effector complex. Genes Dev 30(4):460–470. https://doi.org/10.1101/gad.273722.115.460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Niewoehner O, Garcia-Doval C, Rostøl JT et al (2017) Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers. Nature 548:543

    Article  CAS  Google Scholar 

  22. Kazlauskiene M, Kostiuk G, Venclovas Č et al (2017) A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357:605–609. https://doi.org/10.1126/science.aao0100

    Article  CAS  PubMed  Google Scholar 

  23. Rouillon C, Athukoralage JS, Graham S et al (2018) Control of cyclic oligoadenylate synthesis in a type III CRISPR system. Elife 7:1–25. https://doi.org/10.7554/eLife.36734

    Article  Google Scholar 

  24. McMahon SA, Zhu W, Graham S et al (2020) Structure and mechanism of a Type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate. Nat Commun 11:1–11. https://doi.org/10.1038/s41467-019-14222-x

    Article  CAS  Google Scholar 

  25. Jia N, Mo CY, Wang C et al (2019) Type III-A CRISPR-Cas Csm complexes: assembly, periodic RNA cleavage, DNase activity regulation, and autoimmunity. Mol Cell 73:264–277.e5. https://doi.org/10.1016/j.molcel.2018.11.007

    Article  CAS  PubMed  Google Scholar 

  26. Johnson K, Learn BA, Estrella MA, Bailey S (2019) Target sequence requirements of a type III-B CRISPR-Cas immune system. J Biol Chem 294:10290–10299. https://doi.org/10.1074/jbc.RA119.008728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. You L, Ma J, Wang J et al (2019) Structure studies of the CRISPR-Csm complex reveal mechanism of co-transcriptional interference. Cell 176:239–253.e16. https://doi.org/10.1016/j.cell.2018.10.052

    Article  CAS  PubMed  Google Scholar 

  28. Sofos N, Feng M, Stella S et al (2020) Structures of the Cmr-β complex reveal the regulation of the immunity mechanism of type III-B CRISPR-Cas. Mol Cell 79:741–757.e7. https://doi.org/10.1016/j.molcel.2020.07.008

    Article  CAS  PubMed  Google Scholar 

  29. Lillestøl RK, Shah SA, Brügger K et al (2009) CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties. Mol Microbiol 72:259–272. https://doi.org/10.1111/j.1365-2958.2009.06641.x

    Article  CAS  PubMed  Google Scholar 

  30. Hale CR, Zhao P, Olson S et al (2009) RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139:945–956. https://doi.org/10.1016/j.cell.2009.07.040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zebec Z, Zink IA, Kerou M, Schleper C (2016) Efficient CRISPR-mediated post-transcriptional gene silencing in a Hyperthermophilic archaeon using multiplexed crRNA expression. G3 6:3161–3168. https://doi.org/10.1534/g3.116.032482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zebec Z, Manica A, Zhang J et al (2014) CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus. Nucleic Acids Res 42:5280–5288. https://doi.org/10.1093/nar/gku161

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zink IA, Fouqueau T, Tarrason Risa G et al (2020) Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing. RNA Biol 18(3):421–434. https://doi.org/10.1080/15476286.2020.1813411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zink IA, Pfeifer K, Wimmer E et al (2019) CRISPR-mediated gene silencing reveals involvement of the archaeal S-layer in cell division and virus infection. Nat Commun 10:1–14. https://doi.org/10.1038/s41467-019-12745-x

    Article  CAS  Google Scholar 

  35. Bassani F, Zink IA, Pribasnig T et al (2019) Indications for a moonlighting function of translation factor aIF5A in the crenarchaeum Sulfolobus solfataricus. RNA Biol 16:675–685. https://doi.org/10.1080/15476286.2019.1582953

    Article  PubMed  PubMed Central  Google Scholar 

  36. Peng W, Feng M, Feng X et al (2015) An archaeal CRISPR type III-B system exhibiting distinctive RNA targeting features and mediating dual RNA and DNA interference. Nucleic Acids Res 43:406–417. https://doi.org/10.1093/nar/gku1302

    Article  CAS  PubMed  Google Scholar 

  37. Han W, Feng X, She Q (2017) Reverse gyrase functions in genome integrity maintenance by protecting DNA breaks in vivo. Int J Mol Sci 18:1340. https://doi.org/10.3390/ijms18071340

    Article  CAS  PubMed Central  Google Scholar 

  38. Fricke T, Smalakyte D, Lapinski M et al (2020) Targeted RNA knockdown by a type III CRISPR-Cas complex in zebrafish. CRISPR J 3(4):299–313. https://doi.org/10.1089/crispr.2020.0032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Martusewitsch E, Sensen CW, Schleper C (2000) High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements. J Bacteriol 182:2574–2581. https://doi.org/10.1128/JB.182.9.2574-2581.2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wagner M, van Wolferen M, Wagner A et al (2012) Versatile genetic tool box for the crenarchaeote Sulfolobus acidocaldarius. Front Microbiol 3:1–12. https://doi.org/10.3389/fmicb.2012.00214

    Article  Google Scholar 

  41. Zhang J, Rouillon C, Kerou M et al (2012) Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol Cell 45:303–313. https://doi.org/10.1016/j.molcel.2011.12.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Prangishvili DA, Vashakidze RP, Chelidze MG, Gabriadze IY (1985) A restriction endonuclease SuaI from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. FEBS Lett 192:57–60. https://doi.org/10.1016/0014-5793(85)80042-9

    Article  CAS  PubMed  Google Scholar 

  43. Osawa T, Inanaga H, Sato C, Numata T (2015) Crystal structure of the CRISPR-Cas RNA silencing Cmr complex bound to a target analog. Mol Ther 58:1–14. https://doi.org/10.1016/j.molcel.2015.03.018

    Article  CAS  Google Scholar 

  44. Molina R, Sofos N, Montoya G (2020) Structural basis of CRISPR-Cas type III prokaryotic defence systems. Curr Opin Struct Biol 65:119–129. https://doi.org/10.1016/j.sbi.2020.06.010

    Article  CAS  PubMed  Google Scholar 

  45. Manica A, Zebec Z, Steinkellner J, Schleper C (2013) Unexpectedly broad target recognition of the CRISPR-mediated virus defence system in the archaeon Sulfolobus solfataricus. Nucleic Acids Res 41:10509–10517. https://doi.org/10.1093/nar/gkt767

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Pan S, Li Q, Deng L et al (2019) A seed motif for target RNA capture enables efficient immune defence by a type III-B CRISPR-Cas system. RNA Biol 16:1166–1178. https://doi.org/10.1080/15476286.2019.1618693

    Article  PubMed  PubMed Central  Google Scholar 

  47. Steens JA, Zhu Y, Taylor DW et al (2021) SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation. Nat Commun 12:1–12. https://doi.org/10.1038/s41467-021-25337-5

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Isabelle Anna Zink

    Present address: Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands

Authors and Affiliations

  1. Department of Functional and Evolutionary Ecology, Archaea Biology and Ecogenomics Unit, University of Vienna, Vienna, Austria

    Erika Wimmer & Christa Schleper

Authors
  1. Erika Wimmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Isabelle Anna Zink
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christa Schleper
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christa Schleper .

Editor information

Editors and Affiliations

  1. Cellular Biochemistry of Microorganisms - Biochemie III, Universität Regensburg, Regensburg, Bayern, Germany

    Sébastien Ferreira-Cerca

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Wimmer, E., Zink, I.A., Schleper, C. (2022). Reprogramming CRISPR-Mediated RNA Interference for Silencing of Essential Genes in Sulfolobales. In: Ferreira-Cerca, S. (eds) Archaea. Methods in Molecular Biology, vol 2522. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2445-6_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2445-6_11

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2444-9

  • Online ISBN: 978-1-0716-2445-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation