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

Abstract

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 CRISPR-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 CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization. A bacterial culture is challenged with lytic phages, the surviving cells are screened by PCR for expansion of their CRISPR 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 DNA sequence. The core protocol and its variants serve as a means to explore CRISPR adaptation, discover new CRISPR-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 CRISPR adaptation, this phage-challenge-based approach results in the isolation of CRISPR-immune bacteria for downstream characterization and use.

Characterization of CRISPR‐Cas systems in the Ralstonia solanacearum species complex

“…The R. solanacearum CFBP2957 was challenged with phage phiAP1. Bacteriophage‐insensitive mutants (BIMs) were obtained on plates after a 72‐h incubation period (Hynes et al , ), and their CRISPR arrays were analysed (Fig. A,B).…”

CRISPR-Cas Technology In and Out of the Classroom

“…Although data collection on the outcomes of CRISPR-Cas-based CUREs is ongoing, instructors report increased student learning and engagement after implementing CRISPR-Cas-based CUREs using various metrics. [21][22][23][24][25][26] Academic institutions have only recently started using CRISPR-Cas-based CUREs in their coursework; with increased interest in CRISPR-Cas-based CUREs, more rigorous studies on student achievement will undoubtedly help inform future activities at more institutions.…”

“…In some cases, these experiences can be used to generate both laboratory reagents and data as well as data pertaining to student outcomes resulting from the implementation of CUREs. [21][22][23][24][25][26] Given student interest and the increased need to communicate cutting-edge research to undergraduate students, colleges and universities have begun instituting CRISPR-Casrelated CUREs with great success. Part of the reason why CUREs and other forms of course-based research that utilize CRISPR-Cas technologies are successful is due to the current curricula in molecular biology-based laboratory courses at diverse academic institutions, particularly fouryear colleges and universities.…”

“…[22][23][24]28,30,31 Because CUREs that utilize CRISPR-Cas technology are relatively new, there is limited information about how these academic gains translate into student career choices or persistence in scientific (and particularly those involving genetic engineering) fields. However, as the availability of CRISPR-Cas-based CUREs increases, more information regarding the benefits to students will likely become available.…”

“…Students can utilize lytic phage infection models and screen surviving S. thermophilus cultures for changes in their CRISPR locus using PCR and DNA sequencing. 22 The benefit of this multifaceted CURE is that it offers many variations with many possible avenues for future exploration since students are generating valuable bacterial strains with multiple potential uses, and indeed this sort of discovery by research laboratories has been particularly important in for the dairy industry. 22,23,29 A variety of student products from CUREs can therefore contribute to ongoing faculty research programs.…”