RNA-guided CRISPR nuclease Cas9 cannot reliably differentiate between single nucleotide variations (SNVs) of targeted DNA sequences determined by their guide RNA (gRNA): they typically exhibit similar nuclease activities at any of those variations, unless the variation occurs with specific sequence contexts known as protospacer adjacent motifs (PAMs). Our approach, TOP-SECRETS, generates gRNA variants that allow Cas9 ribonucleoproteins (RNPs) to reliably discriminate between healthy and disease-associated SNVs outside of PAMs.