Detection of Spacer Precursors Formed In Vivo During Primed CRISPR Adaptation

Abstract

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA1. Immunity involves \"adaptation,\" the integration of ~30-bp DNA fragments into the CRISPR array as \"spacer\" sequences, and \"interference,\" the targeted degradation of DNA containing a \"protospacer\" sequence mediated by a complex containing a spacer-derived CRISPR RNA (crRNA)1-4. Specificity for targeting interference to protospacers, but not spacers, occurs through recognition of a 3-bp protospacer adjacent motif (PAM)5 by the crRNA-containing complex6. Interference-driven DNA degradation of protospacer-containing DNA can be coupled with \"primed adaptation,\" ill which spacers are acquired from DNA surrounding the targeted protospacer in a bidirectional, orientation-dependent manner2,3,7. Here we construct a robust in vivo model for primed adaptation consisting of an Escherichia coli type I-E CRISPR-Cas \"self-targeting\" locus encoding a crRNA that targets a chromosomal protospacer. We develop a strand-specific, high-throughput-sequencing method for analysis of DNA fragments, \"FragSeq,\" and use this method to detect short fragments derived from DNA surrounding the targeted protospacer. The detected fragments have sequences matching spacers acquired during primed adaptation, contain ~3- to 4-nt overhangs derived from excision of genomic DNA within a PAM, are generated in a bidirectional, orientation-dependent manner relative to the targeted protospacer, require the functional integrity of machinery for interference and adaptation to accumulate, and function as spacer precursors when exogenously introduced into cells by transformation. DNA fragments with a similar structure accumulate in cells undergoing primed adaptation in a type I-F CRISPR-Cas self-targeting system. We propose the DNA fragments detected in this work are products of universal steps of spacer precursor processing in type I CRISPR-Cas systems.