Reconstitution of CRISPR adaptation in vitro and its detection by PCR

Abstract

CRISPR adaptation is the initial step in CRISPR–Cas immunity and involves the acquisition of foreign invading DNA. Acquisition is facilitated by the almost universally conserved proteins Cas1 and Cas2, which form an adaptation complex. The Cas1–Cas2 complex binds fragments of invading DNA, completes final processing, and catalyzes integration into specific host loci called CRISPR arrays. Structural and biochemical studies from reconstituted complexes have provided mechanistic insight into how CRISPR adaptation occurs; however, these studies have been limited to a narrow subset of CRISPR–Cas types and may not be representative of the other types. Here we describe methods for the purification of the type I-F CRISPR adaptation complex (Cas1:Cas2–3) from Pectobacterium atrosepticum, purification of the DNA architectural protein integration host factor (IHF), and a sensitive PCR-based in vitro integration assay. This assay could easily be used to investigate mechanisms of CRISPR adaptation in other CRISPR–Cas systems, including the roles of accessory proteins.