In Vivo analysis of CAS1/2-CAS3 Proteins Interaction during Primed Adaptation

CRISPR-Cas systems are responsible for two different cellular phenomena: CRISPR interference and CRISPR adaptation. CRISPR interference occurs when a protospacer recognized by the spacer segment of CRISPR RNA is destroyed by Cas effectors. CRISPR adaptation is an integration of new spacers into an array, that relays on conserved Cas1-Cas2 protein complex. For Type I CRISPR-Cas system a unique mode of CRISPR adaptation, named primed adaptation, has been described [Datsenko et al., 2012]. In this course the efficiency of new spacer acquisition is stimulated by recognition of target DNA based on the presence of pre-existed spacer. In Esherichia coli type I-E system the ribonucleoprotein surveillance Cascade complex containing crRNA, which is the product of CRISPR array transcription and processing, recognizes the foreign DNA [Jore et al., 2011]. This recognition serves as a signal for engaging of Cas3 protein which unwinds and degrades the target DNA [Westra et al., 2012]. The Cas1-Cas3 complex was shown at some extent facilitates primed adaptation [Dillard et al., 2018]. We for the first time performed chromatin immunoprecipitation procedure with Cas3-specificic or Cas1-specific antibodies to characterize DNA fragments associated with those proteins in vivo. Close interplay between CRISPR-interference and spacer integration during primed adaptation was revealed based on shared patterns on DNA fragments obtained with anti-Cas1 and anti-Cas3 antibodies. We also analyzed direct interaction between Cas1 and Cas3 protein in vivo. For that propose we obtained E.coli strain containing His-tagged Cas1 protein and cas3 gene deletion. Those cells were transformed with plasmid expressing Cas3 Strep tag and subjected to two-stages chromatography. This study was supported by the Russian Foundation for Basic Research grant 18-34-00048 O.M.