CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas pro-teins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Es-cherichia coli is composed of five different Cas pro-teins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5 ′ handle (8 nt) and a 3 ′ handle (21 nt). The spacer part of crRNA directs Cascade to DNA tar-gets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which di-rect it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5 ′ handle disrupted its binding to Cascade and tar-get DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggest-ing that this complex can be engineered for appli-cations in genome editing and opening the way for incorporation of site-specific labels in crRNA