Developing small Cas9 hybrids using molecular modeling

The RNA-guided CRISPR-Cas9 from Streptococcus pyogenes is the best characterized enzyme for gene editing. Its large size, however, precludes it from being packaged together with its single guide (sg)RNA into a single adeno-associated virus, limiting in vivo applications. Here, we developed smaller Cas9 hybrids, made of the PAM interacting domain (PID) of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues, as well as sgRNA cognate hybrids. Molecular modeling revealed that the presence of a sgRNA stabilizes Cas9. Making the D10A mutation to turn Cas9 into a nickase dramatically alters its binding energy to the sgRNA, showing that the approach can identify functionally relevant changes. However, we found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing Cas9/sgRNA hybrids.