| Summary: | Summary: The multi-subunit type I CRISPR-Cas surveillance complex Cascade uses its crRNA to recognize dsDNA targets. Recognition involves DNA unwinding and base-pairing between the crRNA spacer region and a complementary DNA strand, resulting in formation of an R-loop structure. The modular Cascade architecture allows assembly of complexes containing crRNAs with altered spacer lengths that promise increased target specificity in emerging biotechnological applications. Here we produce type I-E Cascade complexes containing crRNAs with up to 57-nt-long spacers. We show that these complexes form R-loops corresponding to the designed target length, even for the longest spacers tested. Furthermore, the complexes can bind their targets with much higher affinity compared with the wild-type form. However, target recognition and the subsequent Cas3-mediated DNA cleavage do not require extended R-loops but already occur for wild-type-sized R-loops. These findings set important limits for specificity improvements of type I CRISPR-Cas systems. : Songailiene et al. show that engineered versions of Cascade with elongated crRNAs can base-pair with target DNA over 57 bp. However, target recognition only requires wild-type-sized base-pairing of ∼30-bp length. These findings define constraints for improving the specificity of these complexes in biotechnological applications. Keywords: Cascade, CRISPR, type I-E, single molecule, crRNA, R-loop, Streptococcus thermophilus, Cse2, Cse1, genome engineering
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