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Abstract
CRISPR-Cas systems provide their prokaryotic hosts with an adaptive and efficient defense mechanism against invading nucleic acids, and are structurally and functionally classified into six types (Type I-VI). Type III CRISPR-Cas systems are distinguished from the other types of systems by their special requirements and features of target interference. However, our knowledge about adaptation by the Type III systems is very limited. To provide a detailed study to determine the specific properties and patterns of adaptation by the Type III systems, we examined the adaptation by the Type III-A system of Streptococcus thermophilus, and the adaptation by the Type II-A system in the same host as a comparison. Unlike Type II systems and some Type I systems, deletion of genes involved in Type III crRNA biogenesis or interference did not disrupt adaptation nor detectably change spacer uptake patterns except those related to counter-selection. No PAM was observed with the Type III system. The lengths of Type III-A spacers were on average longer than the Type II-A spacers: 36 bp. Interestingly, certain regions of plasmids and the host genome were particularly well-sampled during Type III-A, but not Type II-A, spacer uptake. These regions included the single-strand origins of rolling-circle replicating plasmids, rRNA and tRNA encoding clusters, and promoter regions of expressed genes. We also found that the Type III-A system could adapt and protect the cell from a lytic phage. Collectively, this work indicates that the Type III adaptation machinery preferentially targets DNA secondary structures including imperfect hairpins and other partially double-stranded DNAs. Primed adaptation provides CRISPR-Cas systems with an important co-evolutionary strategy to minimize the escaping invading nucleic acids. Primed adaptation by a Type III system had never been reported. In this dissertation, preliminary evidence of Type III primed adaptation was obtained. Moreover, by studying primed adaptation of the Type I-E CRISPR-Cas system in Streptococcus thermophilus, we established a novel method for the future primed adaptation studies, based on natural CRISPR-Cas escaping viruses.