Procedures for Generating CRISPR Mutants with Novel Spacers Acquired from Viruses or Plasmids

Abstract

CRISPR-Cas systems provide immunity in bacteria and archaea against nucleic acids in the form of viral genomes and plasmids, and influence their coevolution. The first main step of CRISPR-Cas activity is the immune adaptation through spacer(s) acquisition into an active CRISPR locus. This step is also mandatory for the final stage of CRISPR-Cas activity, namely interference. This chapter describes general procedures for studying the CRISPR adaptation step, accomplished by producing bacteriophage-insensitive mutants (BIMs) or plasmid-interfering mutants (PIMs) using various spacer acquisition analyses and experiments. Since each bacterial or archaeal species (and even strain) needs specific conditions to optimize the acquisition process, the protocols described below should be thought of as general guidelines and may not be applicable universally, without modification. Because Streptococcus thermophilus was used as the model system in the first published study on novel spacer acquisition and in many studies ever since, the protocols in this chapter describe specific conditions, media, and buffers that have been used with this microorganism. Details for other species will be given when possible, but readers should first evaluate the best growth and storage conditions for each bacterium-foreign element pair (named the procedure settings) and bear in mind the specificity and variability of CRISPR-Cas types and subtypes. Also, we suggest to be mindful of the fact that some CRISPR-Cas systems are not "naturally" active in terms of the ability to acquire novel CRISPR spacers, and that some systems may require specific conditions to induce the CRISPR-Cas activity for spacer acquisition.