Repeat Size Determination by Two Molecular Rulers in the Type I-E CRISPR Array

Prokaryotic adaptive immune systems are composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. These systems adapt to new threats by integrating short nucleic acids, termed spacers, into the CRISPR array. The functional motifs in the repeat and the mechanism by which a constant repeat size is maintained are still elusive. Here, through a series of mutations within the repeat of the CRISPR-Cas type I-E, we identify motifs that are crucial for adaptation and show that they serve as anchor sites for two molecular rulers determining the size of the new repeat. Adaptation products from various repeat mutants support a model in which two motifs in the repeat bind to two different sites in the adaptation complex that are 8 and 16 bp away from the active site. This model significantly extends our understanding of the adaptation process and broadens the scope of its applications. [Display omitted] •Inverted repeats in the type I-E CRISPR-Cas system are essential for adaptation•Each inverted repeat encodes a motif serving as an anchor site for a molecular ruler•These molecular rulers determine the spacer insertion site regardless of the sequence•The findings support a model considering all known steps in spacer adaptation Goren et al. map elements that are essential for adaptation in the E. coli CRISPR-Cas type I-E repeat. Two elements were identified as anchor sites for two molecular rulers that maintain a constant repeat size. Their findings support a comprehensive model for spacer adaptation.