Developing small Cas9 hybrids using molecular modeling

<p>The RNA-guided CRISPR-Cas9 from <i>Streptococcus pyogenes</i> 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 <i>in vivo</i> applications. Here, we developed smaller Cas9 hybrids, made of the PAM interacting domain (PID) of <i>S pyogenes</i> 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 <i>in silico</i> approaches can identify functional changes caused by point mutations but they may not be well suited for designing Cas9/sgRNA hybrids.&nbsp;</p>