Interactions between Clustered Regularly Interspaced Short Palindromic Repeat RNAs and CRISPR associated proteins (CRISPR-Cas) form a RNA guided adaptive immune system in prokaryotes. The Type I-E Cascade (CRISPR-associated complex for antiviral defense) system is native to E. coli. The Type I-E system targets DNA based on the presence of a protospacer adjacent motif (PAM) and is guided by a RNA known as the guide RNA (gRNA). The Type I-E system has been reported to have at least 5 canonical and several non-canonical PAMs. Although PAM recognition is well studied in the Type I-E system, the relationships between spacer composition, PAM sequence, and eventual cleavage efficacy are not well characterized. We developed a high-throughput in vivo assay in E. coli to test the efficacy of thousands of gRNAs with a wide diversity of PAMs and sequence features. We found (i) the Type I-E Cascade PAM recognition is more expansive than previously reported. (ii) PAM positioning appeared precise, with no evidence for tolerance to PAM slippage. (iii) While a minimal GC-content in the spacer (approximately 50%) was associated with high cleavage efficiency, higher GC-contents didn't seem to further enhance efficiency. (iv) A G at the first position of the guide RNA proximal to the target was associated with higher efficacy of cleavage, and (iv) UUU sequences in the gRNA were associated with lower efficacy. Our findings provide a comprehensive functional profile of the Cascade Type I-E interference gRNA and spacer cleavage requirements.