Development of potent and safe CRISPR tools for in vivo gene editing using directed evolution - Development of potent and safe CRISPR tools for in vivo gene editing using directed evolution Abstract Genome editing, or the ability to precisely manipulate DNA, is an emerging technology that has the potential to be a permanent cure for deadly and debilitating genetic diseases. The development of CRISPR- Cas9 as a genome editing tool has catapulted this field from primary research to clinical trials within the past decade. CRISPR has already been successfully used to treat multiple diseases in human clinical trials, including hereditary blindness, neurodegeneration, blood disorders, and cancer. As these treatments grow in complexity and enter more patients, we increasingly need advanced CRISPR-Cas9 tools that enable potent gene correction while improving safety from aberrant toxic and immunogenic effects. Cas9 is favored for many genome editing approaches due to the ease of reprogramming; the CRISPR single guide RNA (sgRNA) dictates target site through base pairing to a user-defined sequence. However, eukaryotic cells display robust cellular and immunogenic responses towards RNAs, leading to sgRNA instability and toxicity. Chemical RNA modification overcomes this issue by protecting the oligonucleotide from cellular RNA-recognizing machinery, leading to improved stability, distribution, cellular uptake, and safety. Importantly, full chemical modification (modification at every residue) has been essential for the therapeutic success of established RNA therapeutics, like silencing RNA (siRNA). Despite the promises of full chemical modification, fully modified sgRNA remain poorly active for Cas9 genome editing, likely due to the complex interactions between the Cas9 and sgRNA being incompatible with chemical modification. This proposal describes an approach to engineer Cas9 proteins towards compatibility with fully chemically modified sgRNA. These Cas9:sgRNA pairs would enable highly efficient, easy to deliver, and immune evasive genome editing, supporting genome editing applications including multiplexed targets, transient editing, redosing, and vector inactivation.