Extended Data Fig. 1: The leader-repeat stem-loop from the CRISPR-Cas9 system native to Streptococcus pyogenes SF370.
From: Spacer prioritization in CRISPR–Cas9 immunity is enabled by the leader RNA
Accession #: NC_002737.2. a, Array sequence and context within the CRISPR-Cas system. Repeats are in gray, spacers match the corresponding color in the cartoon, and mutations to the consensus repeat are shown in red. The underlined sequence encodes the transcribed RNA leader as determined in S. pyogenes SF37018. The bold and italicized sequence is the putative -10 promoter element, while the lowercase letters designate the stop codon of csn2. The red box indicates the mapped transcriptional start site in E. coli determined using 5′ RACE. b, PCR product generated by 5′ RACE. Biological duplicates are shown. M: DNA marker. C, Predicted minimal free-energy structure of the native and mutated leader-repeat RNA predicted by NUPACK. Left: nucleotide (nt) identities. Right: base-pairing probabilities. d, In vitro determination of the secondary structure and RNase III cleavage sites for the leader-repeat RNA associated with SpyCas9. The transcription start site was extended by 17 nts using the sequence from S. pyogenes to allow visualization of shorter RNAs. Vertical bars: unstructured regions. C: full-length (untreated) 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. Dark and light red arrows indicate the most and second most preferred sites of RNase III cleavage, respectively. Results are representative of triplicate independent experiments. e, 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 within one nt of the equivalent site within the crRNA:tracrRNA duplex (see Fig. 1c). R1: first repeat. S1: first spacer.
