Title: Expanding Cas9 capabilities using synthetic directed evolution
Abstract: CRISPR, bacterial acquired immunity, is an important tool for gene editing across many species. SpCas9, the endonuclease used in this process, snips dsDNA 3 bp upstream of a specific three-nucleotide protospacer adjacent motif (PAM) sequence. Crystal structures of Cas9 protein show that the C-terminal domain of Cas9 plays an important role in PAM binding. Recent efforts to engineer and evolve Cas9 to bind to an arbitrary PAM sequence by mutating the C-terminal domain have shown promising results. Using OrthoRep, an orthogonal replication system in yeast, we can evolve bespoke versions of Cas9 to bind to specific targets of biomedical significance. So far, we have created a yeast strain that shows improved growth when Cas9 is bound to a specific target. These evolved Cas9s will recognize a20 nucleotide target as well as an adjacent 3 nucleotide PAM sequence. Using this technique, we can evolve Base Editors (BE) that will recognize the given target and adjacent PAM but instead of creating double stranded breaks, will nick the required strand and correct the appropriate base. We can use OrthoRep to reduce off-target activity in yeast that can then be verified in mammalian cells. Experimental data will give us a set of mutations that will aid in target binding, and these data can be used to predict the optimal Cas9 for any given sequence using machine learning techniques