A major drawback of CRISPR-Cas9-mediated genome editing is that not all guide RNAs (gRNAs) efficiently cleave target DNA. Although the heterogeneity of gRNA activity is well recognized, the exact way in which CRISPR-Cas9 activity is regulated is not fully understood. In a recent study, a team of researchers used an energy-based model to identify the mechanisms regulating CRISPR-Cas9 activity and specificity. The team investigated the binding free energy, Δ G H , of RNA-DNA interactions and the effects of energy changes during RNA-DNA hybridisation on the efficiency of 11,602 Cas9 gRNAs experimentally validated in other studies. The efficiency of the gRNAs was measured as indel frequency, which is the most accurate indicator of CRISPR-Cas9 activity. They found that Cas9 ‘slippage’ or binding competition on adjacent overlapping protospacer motifs (PAMs) affected gRNA activity by regulating local RNA-DNA interactions. Furthermore, the binding free energy sweet spot quantitatively defines the optimal activity of CRISPR-Cas9 at target and off-target sites in that the gRNA does not bind too weakly or too strongly. In the future, the team will continue to improve their methods, which will include further optimization of the gRNA design for increased efficiency and specificity.