Detecting natural adaptation of the Streptococcus thermophilus CRISPR-Cas systems in research and classroom settings

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRCRISPRSPRSPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRCRISPRSPRSPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRCRISPRSPRSPR immunization. A bacterial culture is challenged with lytic phages, the surviving cells are screened by PCRPCRPCR for expansion of their CRCRISPRSPRSPR array and the newly acquired specificities are mapped to the genome of the phage. Furthermore, we offer three variants of the assay to (i) promote adaptation by challenging the system using defective viruses, (ii) challenge the system using plasmids to generate plasmid-resistant strains and (iii) bias the system to obtain natural immunity against a specifically targeted DNANA sequence. The core protocol and its variants serve as a means to explore CRCRISPRSPRSPR adaptation, discover new CRCRISPRSPRSPR-Cas systems and generate bacterial strains that are resistant to phages or refractory to undesired genes or plasmids. In addition, the core protocol has served in teaching laboratories at the undergraduate level, demonstrating both its robust nature and educational value. Carrying out the core protocol takes 4 h of hands-on time over 7 d. Unlike sequence-based methods for detecting natural CRCRISPRSPRSPR adaptation, this phage-challenge-based approach results in the isolation of CRCRISPRSPRSPR-immune bacteria for downstream characterization and use.