Analysis of Spacers Precursors in Cells Undergoing Naive Adaptation

During primed CRISPR adaptation, spacers precursors of proper length are generated by the Cas3 nuclease-helicase, a component of CRISPR interference machinery that degrades targets recognized by CRISPR effector complex [1]. Naïve adaptation occurs in cells that do not contain spacers matching a target and is critical for initial acquisition of immunity [2,3]. Several studies in Escherichia coli suggest that there is a functional link between the RecBCD recombination complex and Chi DNA sequences it recognizes and spacer acquisition during naïve adaptation [4,5]. DNA fragments generated by RecBCD are thought to be incorporated, at low frequency, into CRISPR array by the Cas1-Cas2 adaptation complex as spacers. The mechanism by which these fragments are selected and processed for incorporation into CRIPR array is not fully understood. To address this issue, we have performed ChIP-Sequencing of Escherichia coli cells undergoing naïve adaptation to reveal DNA fragments associated with the Cas1-Cas2 complex. Our preliminary data suggest that Cas1-Cas2 associated precursor fragments in cells undergoing naïve adaptation are much longer than spacer-sized fragments observed in cells undergoing primed adaptation. A direct correlation between abundance of precursor fragments and number of spacers acquired from them is observed. Further analysis using recBCD mutants will reveal the mechanism by which the precursors of spacers acquired during naïve adaptation are generated.

[1] Musharova, O. Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation. NAR 2017, 45(6), 3297–3307.

[2] Sternberg SH. Adaptation in CRISPR-Cas Systems. Mol Cell. 2016 Mar 17;61(6).

[3] Datsenko KA. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat Commun 2012; 3:945.

[4] Levy, A. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 2015, 520(7548), 505–510.

[5] Ivančić-Bace, I. Different genome stability proteins underpin primed and Naïve adaptation in E. Coli CRISPR-Cas immunity. NAR 2015 43(22).