Abstract
The research objective of this work was to investigate and study details of the CRISPR adaptation process in several different CRISPR-Cas systems as well as use genome-wide CRISPR screening to elucidate cell-intermedilysin interactions. The results of this study showed that Cas1-Cas2 complex from S. thermophilus CRISPR4-Cas system forms a complex that integrates prespacers into the CRISPR array. DnaQ domain fused to Cas2 in this system is a 3’–5’ DNA exonuclease. DnaQ domain is dispensable for spacer integration; however, it serves to trim back overextended 3’ overhangs of the prespacer. Cas1, Cas2, and Csn2 proteins from S. thermophilus CRISPR3-Cas system form at least three different complexes, which interact with Cas9 from the same system via the DNA tether. The identified complexes from CRISPR3-Cas system represent a spacer capture step of the new spacer acquisition process, as they harbour spacer length DNA in their assemblies. Genome-wide CRISPR screening can reveal novel fundamental biological pathways in the membrane composition and lipid metabolism when used in concert with membrane targeting toxins. Intermedilysin has many more dependency factors than previously known CD59 and cholesterol. Among them are heparan sulfates, glucosylceramides, and many other protein or lipid glycosylation factors. ILY can be inhibited by heparin or the removal of heparan sulfates from cells using bacterial heparinases.
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