CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and their associated genes (cas) provide an adaptive immunity against exogenous nucleic acids in bacteria and archaea [1]. During the adaptation a recognition of a short motif known as a PAM (protospacer-adjacent motif) initiates the binding of adaptation complex to a target DNA. Adaptation complex cleaves out a segment of the target DNA and generates a protospacer. After duplication of the repeat, protospacer is inserted into the leader end of the CRISPR array and becomes a spacer [2]. Therefore, the main purpose of this research was to investigate how the sequence of a protospacer affects the adaptation process.
In this study the adaptation complex was co-expressed in E. coli BL21(DE3) cells and purified using a two-step affinity chromatography. Afterwards, in vitro integration assay was carried out. In total 7 different protospacers were analysed. All sequences of protospacers contained an identical segment for the integration into CRISPR array. However, they differed from one another at their 5' and/or 3' ends.
Here we purified a functional adaptation complex and showed that the sequence of a protospacer has an impact for the interaction with adaptation complex. Protospacer that contained sticky ends and double-stranded PAM had the best integration efficiency, which indicates a stronger interaction with adaptation complex.
Taking everything into account, these results provide insight into the mechanism of adaptation complex and protospacer interaction affecting the process of adaptation.