Enabling Focused Ultrasound-Activated Gene Editing with Thermo-Controlled CRISPR-Cas9

PROJECT SUMMARY CRISPR-based therapeutics hold tremendous promise for treating genetic diseases, one of the major final frontiers of medicine. Their ability to edit or correct genetic mutations provides the basis for many future gene- targeted drugs. However, their broad clinical utility in patients is handicapped by two significant challenges – targeted and precise delivery to the correct tissues and organs in the body and reduction of off-target editing, which has the potential to cause new diseases like cancer. Approaches to address in vivo delivery of CRISPR-based therapeutics have focused on searching for delivery vehicles like engineered adeno-associated viruses (AAVs) or lipid nanoparticle (LNP) formulations that can exhibit preference for certain tissues. Specificity has been addressed through bioinformatics approaches that can predict guide RNAs and targets with the lowest off-target potential or engineering of CRISPR-Cas proteins with higher specificity. However, no delivery vehicle, currently or expected in the near term, can exhibit pure tissue-specificity and some organs, like the liver, will invariably be exposed during systemic in vivo delivery. Likewise, off-target editing by CRISPR-Cas enzymes like Cas9 will always be present as prediction tools are not perfect due to the intrinsic nature of the enzyme and natural diversity in human genomes. These shortcomings suggest the need for a radical new approach to targeted delivery and safer gene editing. We propose that combining focused ultrasound (FUS), an established and non-invasive method to perform manipulations deep inside the human body with high resolution in space and time, with gene editing would offer a simple but revolutionary approach to safer targeted gene editing in the future. To accomplish such a lofty goal requires inventing a gene editing technology that can respond to FUS. To accomplish this, we propose a proof-of-concept study to establish feasibility. We hypothesize that the industry-standard Cas9 can be fused to an inhibitory anti-CRISPR (Acr) protein, which will keep the enzyme inactive, via a temperature-sensing protein domain tuned to a thermal response window. Our rationale is that protein folding is naturally cooperative, temperature-responsive protein domains are known and can be thermally tuned, Cas9 can be effectively inhibited by Acrs, and FUS combined with magnetic resonance imaging (MRI) is a mature clinical technology that can control temperature very precisely in deep tissues, such as the brain, in the human body. Our first aim rationally builds several thermo-controlled Cas9s (tc-Cas9s) from existing modular parts, computationally refines those designs, builds them, and tests their properties and activity in vitro and in cells. Our second aim creates randomized mutagenesis libraries and uses thermal shifts in cells to perform directed evolution and select tc-Cas9s that perform editing in the desired temperature regimes. Together, these results will test the feasibility of creating gene editing platforms that can be controlled non-invasively with FUS, potentially rethinking delivery and safety for therapeutic gene editing.