Conformational Control of Cascade Interference and Priming Activities in CRISPR Immunity

Abstract

Summary During Type I-E CRISPR-Cas immunity, the Cascade surveillance complex utilizes CRISPR-derived RNAs to target complementary invasive DNA for destruction. When invader mutation blocks this interference activity, Cascade instead triggers rapid primed adaptation against the invader. The molecular basis for this dual Cascade activity is poorly understood. Here we show that the conformation of the Cse1 subunit controls Cascade activity. Using FRET, we find that Cse1 exists in a dynamic equilibrium between “open” and “closed” conformations, and the extent to which the open conformation is favored directly correlates with the attenuation of interference and relative increase in priming activity upon target mutation. Additionally, the Cse1 L1 motif modulates Cascade activity by stabilizing the closed conformation. L1 mutations promote the open conformation and switch immune response from interference to priming. Our results demonstrate that Cascade conformation controls the functional outcome of target recognition, enabling tunable CRISPR immune response to combat invader evolution.

Real-Time Observation of Target Search by the CRISPR Surveillance Complex Cascade

“…However, this model does not account for how Cascade may interact with DNA prior to helical bending. Interestingly, when the two models are docked to ensure linearity of the DNA, Cse1 adopts a partially “open” conformation, as has been observed in the crystal structure of ssDNA-bound Cascade and through FRET studies of Cse1 conformation (Mulepati et al, 2014; Xue et al, 2016) (Fig. 5C).…”

“…We hypothesized that Cascade may more efficiently form a full R-loop in our smFRET assay if the double-stranded region of the target were truncated, as has previously been shown in bulk biochemical assays (Xue et al, 2016). To test this, we designed a dsDNA target with a truncated non-target strand (dsDNA trunc ) forming only 10 base pairs (bp) with the PAM-proximal region of the target (Fig.…”

“…Using a previously developed fluorescently-labeled Cascade (Xue et al, 2016), we established a system to directly observe individual Cascade complexes binding to a complementary target dsDNA in real time using smFRET (Fig. 1A).…”

“…We interpreted these events as random target sampling or incomplete R-loop formation. Previous bulk biochemical studies have shown that Cascade binds to full length dsDNA oligonucleotides with low affinity at room temperature, which may account for the low percentage of full R-loop formation events (Xue et al, 2016). These results suggest that Cascade requires additional energy to efficiently unwind dsDNA for full R-loop formation, contributed either by thermal energy or negative supercoiling (Westra et al, 2012).…”

“…The Cse1 PAM recognition motif identifies PAMs during target searching and maintains contact following target binding, an interaction that is thought to be important for stabilizing Cse1 in a conformation required for Cas3 recruitment and target degradation (Hayes et al, 2016; Xiao et al, 2017; Xue et al, 2016). In addition, our data also implicate the Cse1 lysine-rich β hairpin in the PAM searching process, although this motif does not contact DNA following target binding.…”