Fig. 5: Event characteristics for Cas9d10a and the Cas9d10a + sgRNA complex under asymmetric salt conditions (1 M inside pore/4 M KCl outside pore).

a Graph of net charge based on calculations of amino acid composition of Cas9d10a alone and Cas9d10a-sgRNA at various pH values. The black dotted line represents the pH value of our working conditions (7.4), and the red and blue circles are the corresponding charges of Cas9d10a + sgRNA and Cas9d10a alone, respectively. b Current trace of Cas9d10a with and without sgRNA in asymmetric salt conditions (1 M KCl inside and 4 M KCl outside). Events were resistive when Cas9d10a was inside the nanopipette, and a positive voltage is applied inside the nanopipette. Cas9d10a was pre-incubated before diluting in 1 M KCl where the Cas9d10a and sgRNA were in an equimolar ratio (1:1). Events were conductive when Cas9d10a + sgRNA was inside the nanopipette and a negative voltage was applied inside the nanopipette. c Schematic of set-up (left) and fluxes of K⁺ and Cl⁻ under both conditions. Top condition includes the negatively charged complex of Cas9d10a and sgRNA. The bottom condition contains the positively charged Cas9d10a molecule alone. Both have 1 M KCl + analyte within the nanopipette and 4 M KCl within the electrolyte bath solution. d Current enhancement observed for both DNA and protein using similar sized pores (36 nS for Cas9:sgRNA and 33 nS for λ-DNA) and the same voltage bias of −500 mV. An asymmetric salt condition was used on both experiments (1 M/4 M KCl) and voltage was applied inside the nanopipette; driving negative DNA and protein–RNA complexes out of the nanopipette.