1 Determining the Specificity of Cascade Binding , Interference , and Priming in vivo 2 3 4 5

34 35 In CRISPR immunity systems, short CRISPR RNAs (crRNAs) are bound by CRISPR-associated 36 (Cas) proteins, and these complexes target invading nucleic acid molecules for degradation in a 37 process known as interference. In Type I CRISPR systems, the Cas protein complex that binds 38 DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition 39 of new immunity elements, in a process known as priming. The sequence determinants for 40 protospacer binding and interference have been well characterized for Type II CRISPR systems 41 such as the Cas9 system of Streptococcus pyogenes. In contrast, relatively little is known about 42 the requirements for Cascade-DNA binding, interference, and priming in Type I systems. Here, 43 we use genome-scale approaches to assess the specificity determinants for Cascade-DNA 44 interaction, interference, and priming in vivo for the Type I-E system of Escherichia coli. 45 Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a 46 DNA target. Consequently, a single crRNA promotes Cascade association with numerous off47 target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal 48 sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for 49 both interference and priming. Hence, Cascade binding to sub-optimal, off-target sites is inert. 50 Our data support a model in which initial Cascade association with DNA targets requires little 51 sequence complementarity at the crRNA 5 end, whereas recruitment and/or activation of the 52 Cas3 nuclease, a prerequisite for interference and priming, requires extensive base-pairing. 53 54 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/169011 doi: bioRxiv preprint first posted online Jul. 27, 2017;