Fig. 2. Stimuli-responsive gRNA for conditional control of CRISPR-Cas9 function in vitro. (A) CRISPR-Cas9 complex containing Cas9 (gray), PS-2′-OMe-modified crRNAEMX1 (blue) targeting the human EMX1 DNA, tracrRNA (purple), and substrate DNA (green) with a PAM (black). The red arrows and red nucleotides indicate PS-2′-OMe sites in crRNAEMX1 used for BO and CM modifications. Inset is the unwound human EMX1 locus targeted by the PS-2′-OMe-crRNAEMX1 containing 3 PS-2′-OMe sites. The 10-nt seed region is underlined. Am* and Gm* in red are PS-2′-OMe-modified nucleotides. (B) Removal of BO from 3BO-141718-crRNAEMX1 by H2O2 at different concentrations. Reactions were allowed to proceed for 1 h at 37 °C. (C) Cleavage of double-stranded EMX1 DNA substrate by unmodified and modified crRNAs with or without H2O2 treatment. From left to right: lane 1–3, DNA only, DNA + crRNAEMX1, DNA + 3PS-2′-OMe-141718-crRNAEMX1; lane 4–11, DNA + 3BO-141718-crRNAEMX1 treated with H2O2 at indicated concentrations. “M” is the ladder lane containing DNA standards of 100, 250, 500, 750, 1000, and 2000 bp. Cas9 and tracrRNA were supplied in all the reactions. (D) Time-dependent removal of CM from 3CM-141718-crRNAEMX1 when irradiated by visible light at 470 nm (top) or 405 nm (bottom). (E) Cleavage of double-stranded EMX1 DNA substrate promoted by 3PS-2′-OMe-141718-crRNAEMX1 and 3CM-141718-crRNAEMX1 with different duration of light irradiation. Cas9 and tracrRNA were supplied in all the reactions.
