Abstract
In Escherichia coli, acquisition of new spacers in the course of CRISPR-Cas adaptation is dramatically stimulated by preexisting partial matches between a bacterial CRISPR cassette spacer and a protospacer sequence in the DNA of the infecting bacteriophage or plasmid. This phenomenon, which we refer to as “priming,” can be used for very simple and rapid construction of multiple E. coli strains capable of targeting, through CRISPR interference, any phage or plasmid of interest. Availability of such strains should allow rapid progress in the analysis of CRISPR-Cas system function against diverse mobile genetic elements.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pul U, Wurm R, Arslan Z, Geissen R, Hofmann N, Wagner R (2010) Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol Microbiol 75:1495–1512
Pougach K, Semenova E, Bogdanova E, Datsenko KA, Djordjevic M, Wanner BL, Severinov K (2010) Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol Microbiol 77:1367–1379
Westra ER, Pul U, Heidrich N, Jore MM, Lundgren M, Stratmann T, Wurm R, Raine A, Mescher M, Van Heereveld L, Mastop M, Wagner EG, Schnetz K, Van Der Oost J, Wagner R, Brouns SJ (2010) H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Mol Microbiol 77:1380–1393
Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321:960–964
Datsenko KA, Pougach K, Tikhonov A, Wanner BL, Severinov K, Semenova E (2012) Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun 3:945
Semenova E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, Severinov K (2011) Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A 108:10098–10103
Shmakov S, Savitskaya E, Semenova E, Datsenko KA, Severinov K (2014) Pervasive generation of oppositely-oriented spacers during CRISPR adaptation. Nucleic Acids Res 42:5907–5916
Lee SY, Rasheed S (1990) A simple procedure for maximum yield of high-quality plasmid DNA. Biotechniques 9:676–679
Acknowledgments
This work was supported by National Institutes of Health grant R01 GM10407, Molecular and Cell Biology Program grant from the Russian Academy of Sciences Presidium, and Ministry of Education and Science of Russian Federation project 14.B25.31.0004 and Russian Science Foundation grant 14-14-00988 (to KS) and Russian Foundation for Basic Research grant 14-04-00916 (to Ekaterina Savitskaya).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Strotksaya, A., Semenova, E., Savitskaya, E., Severinov, K. (2015). Rapid Multiplex Creation of Escherichia coli Strains Capable of Interfering with Phage Infection Through CRISPR. In: Lundgren, M., Charpentier, E., Fineran, P. (eds) CRISPR. Methods in Molecular Biology, vol 1311. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2687-9_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2687-9_9
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2686-2
Online ISBN: 978-1-4939-2687-9
eBook Packages: Springer Protocols