Sarah, Russell Sage College
Mentor: Joseph Wade
CRISPR, or clustered regularly interspaced short palindromic repeats, is an adaptive immune system found in roughly 50% of bacteria and 90% of archaea. This system works through three stages: adaptation, biogenesis, and interference. Acquisition of new CRISPR immunity elements, known as spacers, occurs during adaptation. There are two types of spacer acquisition: naïve and priming, both of which require different protein elements. Naïve acquisition requires Cas, or CRISPR associated genes 1 and 2, whereas priming adaptation, the focus of this project, requires Cas 3. A variety of elements are necessary to make adaptation function; however, these requirements have only recently been explored.
Integration host factor, or IHF, has recently been identified as a protein element necessary for naïve adaptation (Nunez et al., 2016). In this study we investigated if IHF plays role in priming spacer acquisition, and explored Hu as a potential protein element necessary to adaptation, as Hu is a homologue of IHF.
Additionally, recent studies have shown that the leader sequence is a necessary genetic element for spacer acquisition (Yosef et al., 2012). The leader sequence is a region of roughly 150 base pairs that separates the CRISPR array and Cas genes. A study performed in 2012 showed that the 40-60 bp region upstream from the first repeat in the CRISPR I array is important to spacer acquisition (Yosef et al.). In this project, 10 bp chunks were deleted from the 40-60 bp region via FRUIT, or flexible recombineering using the integration of ThyA. A region conserved across many Type I-E CRISPR systems was also deactivated through point mutations introduced via FRUIT. Priming assay results suggest that IHF and all three leader sequence elements described are necessary for priming spacer acquisition, while Hu is not.