TY - JOUR T1 - Determining the Specificity of Cascade Binding, Interference, and Priming <em>in vivo</em> JF - bioRxiv DO - 10.1101/169011 SP - 169011 AU - Lauren A. Cooper AU - Anne M. Stringer AU - Joseph T. Wade Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/07/27/169011.abstract N2 - SIGNIFICANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61 nt CRISPR RNAs (crRNAs). The Cascade-crRNA complex is directed to invading DNA molecules through base-pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease that destroys the invading DNA molecule, and promotes acquisition of new immunity elements. We show that Cascade-crRNA binding to DNA is highly promiscuous in vivo. Consequently, endogenous E. coli crRNAs direct Cascade binding to &gt;100 chromosomal locations. In contrast, target degradation and acquisition of new immunity elements requires highly specific association of Cascade-crRNA with DNA, limiting CRISPR-Cas function to the intended targets.ABSTRACT In CRISPR 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 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 priming. The sequence determinants for protospacer binding and interference have been well characterized for Type II CRISPR systems such as the Cas9 system of Streptococcus pyogenes. In contrast, relatively little is known about the requirements for Cascade-DNA binding, interference, and priming in Type I systems. Here, we use genome-scale approaches to assess the specificity determinants for Cascade-DNA interaction, interference, and priming 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 &gt;100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, &gt;18 bp are required for both interference and priming. 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 little sequence complementarity at the crRNA 5□ end, whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and priming, requires extensive base-pairing. ER -