Extended Data Fig. 7: In vitro determination of the secondary structure and RNase III cleavage sites for the leader-repeat RNA associated with LrhCas9 and Sth1Cas9.
From: Spacer prioritization in CRISPR–Cas9 immunity is enabled by the leader RNA
a, In vitro determination of the secondary structure and RNase III cleavage sites for the leader-repeat RNA associated with LrhCas9. The probed RNA was 5′ radiolabeled and resolved by denaturing PAGE. The 5′ end was truncated to focus on the predicted secondary structure involving the repeat. Vertical bars on the right indicate unstructured regions. C - full-length control. T1: Ladder of G’s generated by incubating the RNA with RNase T1. OH: single-nucleotide ladder generated by incubating the RNA under basic conditions. RNase III: the RNA was incubated with the indicated units of E. coli RNase III (0, 0.0016, 0.008, 0.04, 0.2, 1) for 5 min at 37 °C. Dark and light red arrows indicate the most preferred and second most preferred sites of RNase III cleavage, respectively. Results are representative of triplicate independent experiments. b, Corresponding secondary structure of the leader-repeat RNA. Circles indicate unstructured bases identified by in-line probing. The preferred site of RNase III cleavage lies below the equivalent site within the crRNA:tracrRNA duplex (see Extended Data Fig. 8a). R1: first repeat. S1: first spacer. c, In vitro determination of the secondary structure and RNase III cleavage sites for the leader-repeat RNA associated with Sth1Cas9. See a for details. The 5′ end was truncated to focus on the predicted secondary structure involving the repeat. Results are representative of triplicate independent experiments. d, Corresponding secondary structure of the leader-repeat RNA. Circles indicate unstructured bases identified by in-line probing. The preferred site of RNase III cleavage lies above the equivalent site within the crRNA:tracrRNA duplex (see Extended Data Fig. 8d). R1: first repeat. S1: first spacer.
