Effect of Cas9 Protein on the Seed-Target Base Pair of the sgRNA/DNA Hybrid Duplex

CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein (Cas9) has been widely used for gene editing. Not all guide RNAs can cleave the DNA efficiently remains a major challenge to CRISPR/Cas9-mediated genome engineering. Therefore, understanding how the Cas9 complex successfully and efficiently identifies specific functional targets through base-pairing has great implications for such applications. The 10-nt seed sequence at the 3' end of the guide RNA is critical to target recognition and cleavage. Here, through stretching molecular dynamics simulation, we studied the thermodynamics and kinetics of the binding-dissociation process of the seed base and the target DNA base with the Cas9 protein. The results showed that in the presence of Cas9 protein, the enthalpy change and entropy change in binding-dissociation of the seed base with the target are smaller than those without the Cas9 protein. The reduction of entropy penalty upon association with the protein resulted from the pre-organization of the seed base in an A-form helix, and the reduction of enthalpy change was due to the electrostatic attraction of the positively charged channel with the negative target DNA. The binding barrier coming from the entropy loss and the dissociation barrier resulting from the destruction of the base pair in the presence of Cas9 protein were lower than those without protein, which indicates that the seed region is crucial for efficiently searching the correct target by accelerating the binding rate and dissociating fast from the wrong target.