title: CRISPR-based screening of genomic island excision events in bacteria.
abstract: Genomic analysis of Streptococcus thermophilus revealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats-CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPR-Cas systems and MGEs is one of the driving forces governing genome homeostasis in this genus. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs, in silico prediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. Targeting lacZ within the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac(-) survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.
authors.affiliation: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695; Functional Genomics Graduate Program, North Carolina State University, Raleigh, NC 27695.
authors.forename: Kurt
authors.initials: K
authors.lastname: Selle
authors.affiliation: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695; Functional Genomics Graduate Program, North Carolina State University, Raleigh, NC 27695 klaenhammer@ncsu.edu rbarran@ncsu.edu.
authors.forename: Todd R
authors.initials: TR
authors.lastname: Klaenhammer
authors.affiliation: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695; klaenhammer@ncsu.edu rbarran@ncsu.edu.
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Barrangou
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D017353:Gene Deletion; D016680:Genome, Bacterial; D006706:Homeostasis; D059765:Homologous Recombination; D048249:Streptococcus thermophilus / Q000235:genetics* / Q000254:growth & development
keywords: CRISPR; Cas9; IS-elements; lactic acid bacteria; transposons