Fig. 5

Structure and recognition of the single-guide RNA (sgRNA) scaffold. a Structure of the sgRNA scaffold. The guide region is omitted for clarity. The disordered region is shown as a dotted line. b Binding of the sgRNA scaffold to Corynebacterium diphtheriae Cas9 (CdCas9) (stereo view). The disordered region of the sgRNA is shown as a dotted line. c Effects of the sgRNA truncation. The linearized plasmid target with the GGGAAAC protospacer adjacent motif (PAM) was incubated at 37 °C for 0.5 and 5 min with CdCas9 and the truncated CdCas9 sgRNA (22-nt guide) or the Streptococcus pyogenes Cas9 (SpCas9) sgRNA (22-nt guide). 1–112, the full-length sgRNA; ∆57–65, the sgRNA, in which nucleotides 57–65 were replaced with GAAA; ∆87–107, the sgRNA, in which nucleotides 87–107 were replaced with GAAA; 1–81, the sgRNA, in which nucleotides 82–112 were truncated; SpCas9, the SpCas9 sgRNA. d Base-specific recognition of the sgRNA linker region (stereo view). Hydrogen bonds are shown as dashed lines. e Superimposition of the CdCas9 sgRNA and the SpCas9 sgRNA (PDB: 4OO8). The disordered region is shown as a dotted line. Error bars represent s.d. from n = 3 replicates