CRISPR pp 277-291 | Cite as

Cas3 Nuclease–Helicase Activity Assays

  • Tomas Sinkunas
  • Giedrius Gasiunas
  • Virginijus SiksnysEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1311)


Cas3 is a signature protein of the type I CRISPR-Cas systems and typically contains HD phosphohydrolase and Superfamily 2 (SF2) helicase domains. In the type I CRISPR-Cas systems Cas3 functions as a slicer that provides foreign DNA degradation. Biochemical analysis indicate that Cas3 of the Streptococcus thermophilus DGCC7710 (St-Cas3) CRISPR4 system is a single-stranded DNA nuclease which also possesses a single-stranded DNA-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes in 3′ to 5′ direction. The interplay between the nuclease and ATPase/helicase activities of St-Cas3 results in DNA degradation. Here, we describe assays for monitoring of St-Cas3 nuclease, ATPase and helicase activities in a stand-alone form and in the presence of the Cascade ribonucleoprotein complex. These assays can be easily adapted for biochemical analysis of Cas3 proteins from different microorganisms.

Key words

Cas3 CRISPR Cascade Streptococcus thermophilus 



We thank Rodolphe Barrangou and Philippe Horvath for discussions. The work on CRISPR systems in Siksnys’s laboratory was funded by the European Social Fund under Global Grant measures.


  1. 1.
    Barrangou R, Fremaux C, Deveau H et al (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819):1709–1712. doi: 10.1126/science.1138140 CrossRefPubMedGoogle Scholar
  2. 2.
    Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327:167–170, 10.1126/science.1179555CrossRefPubMedGoogle Scholar
  3. 3.
    Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482:331–338. doi: 10.1038/nature10886 CrossRefPubMedGoogle Scholar
  4. 4.
    Gasiunas G, Sinkunas T, Siksnys V (2013) Molecular mechanisms of CRISPR-mediated microbial immunity. Cell Mol Life Sci. doi: 10.1007/s00018-013-1438-6 PubMedCentralGoogle Scholar
  5. 5.
    Makarova KS, Haft DH, Barrangou R et al (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9:467–477. doi: 10.1038/nrmicro2577 CrossRefPubMedGoogle Scholar
  6. 6.
    Sinkunas T, Gasiunas G, Fremaux C et al (2011) Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J 30:1335–1342. doi: 10.1038/emboj.2011.41 CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Sinkunas T, Gasiunas G, Waghmare SP et al (2013) In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus. EMBO J 32:385–394. doi: 10.1038/emboj.2012.352 CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Haft DH, Selengut J, Mongodin EF, Nelson KE (2005) A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol 1:e60. doi: 10.1371/journal.pcbi.0010060 CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Makarova KS, Grishin NV, Shabalina SA et al (2006) A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1:7. doi: 10.1186/1745-6150-1-7 CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Westra ER, van Erp PBG, Künne T et al (2012) CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell 46:595–605. doi: 10.1016/j.molcel.2012.03.018 CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Mulepati S, Bailey S (2011) Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3). J Biol Chem 286:31896–31903. doi: 10.1074/jbc.M111.270017 CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci U S A 74:560–564CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Tomas Sinkunas
    • 1
  • Giedrius Gasiunas
    • 1
  • Virginijus Siksnys
    • 1
    Email author
  1. 1.Institute of BiotechnologyVilnius UniversityVilniusLithuania

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