Date of Award

1-1-2018

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (ii, vii, 258 pages) : illustrations (some color)

Dissertation/Thesis Chair

Joseph T Wade

Committee Members

Keith Derbyshire, Nick Mantis, Nilesh Banavali, Thomas Begley

Keywords

Cascade, CRISPR, CRISPR-Cas, E. coli, Type I-E, Escherichia coli, DNA-binding proteins

Subject Categories

Genetics

Abstract

In CRISPR-Cas immunity systems, short CRISPR RNAs (crRNAs) are bound by CRISPR-associated (Cas) proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, ~24 bp are required for both interference and primed adaptation. Hence, Cascade binding to sub-optimal, off-target sites is inert. Our data support a model in which initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5ʹ end, whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base-pairing.

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