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.
