TOP-SECRETS enables Cas9 nucleases to discriminate SNVs outside of PAMs
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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.
