Extended Data Fig. 8: Nonspecific single-stranded RNA cleavage by TbaIscB.
From: Structural visualization of the molecular evolution of CRISPR–Cas9
(a) In vitro RNA and DNA cleavage activities of WT TbaIscB and its active-site mutants. The TbaIscB protein (WT, dRuvC, or dHNH) was incubated with either the Cy5-labeled single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), or ssDNA substrate (60 nt) at 37 °C for 1 or 5 min, and then the reaction was analyzed by 15% urea-PAGE. dRuvC, D59A; dHNH, H256A. (b) In vitro ssRNA cleavage activities of TbaIscB, HfmIscB, YnpsCas9, and NbaCas9. The Cy5-labeled ssRNA substrate (60 nt) was incubated with either the TbaIscB, YnpsCas9, or NbaCas9 protein, or the HfmIscB–ωRNA complex at 37 °C for 1 or 60 min, and then the reaction was analyzed by 15% urea-PAGE. HfmIscB was only purified as the ribonucleoprotein complex. In (a) and (b), experiments were repeated three times with similar results. (c) Multiple sequence alignment of the HNH domains from TbaIscB, NmCas9, CjCas9, OgeuIscB, HfmIscB, YnpsCas9, and NbaCas9. Key residues for RNA cleavage are highlighted in red. (d) Structural comparison of the HNH domains of TbaIscB (AlphaFold2 model) and NmCas9 (PDB: 8JA0).
