Transcriptional Knockdown in Pneumococci Using CRISPR Interference

  • Morten KjosEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1968)


Sequence-specific knockdown of gene expression using CRISPR interference (CRISPRi) has recently been developed for Streptococcus pneumoniae. By coexpression of a catalytically inactive Cas9-protein (dCas9) and a single guide RNA (sgRNA), CRISPRi can be used to knock down transcription of any gene of interest. Gene specificity is mediated by a 20 bp sequence on the sgRNA, and new genes can be targeted by replacing this 20 bp sequence. Here, a protocol is provided for design of sgRNAs and construction of CRIPSRi strains in S. pneumoniae, based on the vectors published by Liu et al. (Mol Syst Biol 13:931, 2017).

Key words

CRISPRi dCas9 Knockdown sgRNA Inverse PCR 


  1. 1.
    Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183CrossRefGoogle Scholar
  2. 2.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefGoogle Scholar
  3. 3.
    Liu X, Gallay C, Kjos M, Domenech A, Slager J, van Kessel SP, Knoops K, Sorg RA, Zhang JR, Veening JW (2017) High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Mol Syst Biol 13:931CrossRefGoogle Scholar
  4. 4.
    Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS (2013) CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc 8:2180–2196CrossRefGoogle Scholar
  5. 5.
    Peters JM, Colavin A, Shi H, Czarny TL, Larson MH, Wong S, Hawkins JS, Lu CH, Koo BM, Marta E et al (2016) A comprehensive, CRISPR-based functional analysis of essential genes in bacteria. Cell 165:1493–1506CrossRefGoogle Scholar
  6. 6.
    Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA (2013) Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res 41:7429–7437CrossRefGoogle Scholar
  7. 7.
    Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239CrossRefGoogle Scholar
  8. 8.
    van Raaphorst R, Kjos M, Veening JW (2017) Chromosome segregation drives division site selection in Streptococcus pneumoniae. Proc Natl Acad Sci U S A 114:E5959–E5968CrossRefGoogle Scholar
  9. 9.
    Martin B, Garcia P, Castanie MP, Claverys JP (1995) The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction. Mol Microbiol 15:367–379CrossRefGoogle Scholar
  10. 10.
    Lacks S, Hotchkiss RD (1960) A study of the genetic material determining an enzyme in pneumococcus. Biochim Biophys Acta 39:508–518CrossRefGoogle Scholar
  11. 11.
    Hawkins JS, Wong S, Peters JM, Almeida R, Qi LS (2015) Targeted transcriptional repression in bacteria using CRISPR interference (CRISPRi). Methods Mol Biol 1311:349–362CrossRefGoogle Scholar
  12. 12.
    Lorenz R, Bernhart SH, Honer Zu Siederdissen C, Tafer H, Flamm C, Stadler PF, Hofacker IL (2011) ViennaRNA Package 2.0. Algorithms Mol Biol 6:26CrossRefGoogle Scholar
  13. 13.
    Paik J, Kern I, Lurz R, Hakenbeck R (1999) Mutational analysis of the Streptococcus pneumoniae bimodular class A penicillin-binding proteins. J Bacteriol 181:3852–3856PubMedPubMedCentralGoogle Scholar
  14. 14.
    Hoskins J, Matsushima P, Mullen DL, Tang J, Zhao G, Meier TI, Nicas TI, Jaskunas SR (1999) Gene disruption studies of penicillin-binding proteins 1a, 1b, and 2a in Streptococcus pneumoniae. J Bacteriol 181:6552–6555PubMedPubMedCentralGoogle Scholar
  15. 15.
    Sambrook JF, Russell DW (2001) Molecular cloning: a laboratory manual, vol 1,2 and 3, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  16. 16.
    Sorg RA, Kuipers OP, Veening JW (2015) Gene expression platform for synthetic biology in the human pathogen Streptococcus pneumoniae. ACS Synth Biol 4:228–239CrossRefGoogle Scholar
  17. 17.
    Sorg RA (2016) Engineering approaches to investigate pneumococcal gene expression regulation and antibiotic resistance development. Ph.D. thesis, University of GroningenGoogle Scholar
  18. 18.
    Yan M, Zhou SR, Xue HW (2015) CRISPR primer designer: design primers for knockout and chromosome imaging CRISPR-Cas system. J Integr Plant Biol 57:613–617CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Faculty of Chemistry, Biotechnology and Food ScienceNorwegian University of Life SciencesÅsNorway

Personalised recommendations