CRISPR-Chip Formation and Activation

After PBA linker immobilization and activation on the surface of the graphene, 900 ng (30 μl in 2 mM MgCl₂ of dCas9 (University of California, Berkeley MacroLab) was incubated for 15 min at 37 °C on the surface of the graphene. The CRISPR–Chip I response was monitored continuously. Unreacted PBA molecules on the graphene surface were then blocked using amino-PEG5-alcohol (1 mM, 10 min at 37 °C) and ethanolamine hydrochloride (1 M, 10 min at 37 °C) (Sigma–Aldrich). After blocking, the graphene surface was washed with 2 mM MgCl₂ solution and incubated until the I response readings stabilized. To form the dRNP complex, 900 ng (30 μl in 2 mM MgCl₂ of sgRNA specific to the target sequence was introduced onto the graphene surface and incubated for 10 min at 37 °C to form dRNPs. sgRNA samples were thermally treated to remove the dimer structure before incubation with the CRISPR–Chip76 (link). The CRISPR–Chip was then washed with 2 mM MgCl₂ for 5 min to remove any unbound sgRNA. This final step resulted in full dRNP formation on the graphene surface and a functional CRISPR–Chip.