Almost all archaea and half of bacteria contain Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins (CRISPR-Cas) adaptive immune systems that protect them against foreign genetic elements invasion. While diverse, all CRISPR-Cas function through three common steps: (i) adaptation, i.e., acquisition of foreign DNA segments (spacers) into CRISPR arrays; (ii) CRISPR array transcription and transcript processing to produce mature CRISPR RNAs (crRNAs), and (iii) interference, when Cas effector enzymes are guided by crRNAs to matching targets leading to target cleavage and ultimate invader genome destruction. As any immune system, CRISPR-Cas must be capable of self/non-self discrimination to prevent autoimmune death of the host caused by acquisition of spacers from own DNA followed by self-interference. A remarkable mechanism of self/non-self discrimination called ?priming? operates in type I CRISPR-Cas systems: acquisition of spacers from DNA containing partial matches to pre-existing CRISPR array spacers is dramatically stimulated compared to acquisition from DNA devoid of such sequences. While partially matching crRNAs that promote primed adaptation are incapable of efficient interference, all components of the interference machinery are required for primed adaptation. The mechanistic relationship between the interference and adaptation modules of CRISPR-Cas response during primed adaptation is not clear. In this work, protein-nucleic acid complexes and nucleic acid intermediates of CRISPR interference and primed adaptation by the Type I CRISPR- Cas system of Escherichia coli, the best-studied microbe, will be characterized in vivo and in vitro, and host functions that affect both processes will be revealed. In addition to uncovering a functional link between CRISPR interference and primed adaptation, programmable effector complexes with expanded targeting potential will be created as a result of proposed work and highly sensitive quantitative biophysical methods to study effector-target interactions will be developed.
CRISPR-Cas systems of bacterial immunity against viruses are revolutionizing human genetic medicine but also present profound questions to basic sciences - microbiology, molecular biology and evolution. We will study how these systems respond in different way to viruses: either destroying them outright or acquiring additional fragments of viral DNA to achieve even higher levels of immunity.
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