SUMMARY There is an unmet need for delivery systems that empower broad therapeutic use of gene editing technologies. The power of the CRISPR/Cas system has been harnessed for a variety of gene editing approaches, and it has been hailed as the future of therapeutic gene editing. A key impediment to the therapeutic implementation of CRISPR/Cas-mediated gene editing strategies is the efficient delivery of the required components, i.e., two non- coding RNAs and the large Cas9 nuclease, to target cells and tissues. Current delivery platforms, e.g., viruses and non-viral platforms, suffer from several weaknesses, including (1) lack of targeting specificity, 2) inability to enter cells, 3) immune activation, 4) off-target effects and limited therapeutic window, and 5) limited genetic encoding and cargo capacity. SiVEC Biotechnologies, with support from phase I and phase II SBIR grants from NIAID (1R43AI140243-01A1 and 2R44AI140243-02), has previously developed a bacteria-based delivery platform to efficiently generate and deliver short-hairpin RNAs (shRNAs) to specifically targeted tissues. This platform is not limited to shRNA delivery, and in this proposal, we will engineer it to create “SiCRISP” – a novel platform that produces and delivers all components needed for CRISPR/Cas9-directed gene editing. We will also perform proof-of-concept studies for its use in the delivery of gene editing machinery to clinically relevant tissues. By applying the key components of the SiVEC delivery platform, SiCRISP overcomes several critical shortcomings that limit the therapeutic potential of existing gene editing delivery systems: (1) it can target specific cells and tissues, and can be administered via multiple routes, e.g., intranasal, systemic, targeted injection, intraocular, intravaginal, among others; (2) it can enter cells and escape the endosome to efficiently deliver its cargo; (3) it has been experimentally confirmed as non-immunogenic, and it is not recognized by host immune cells, even after repeated delivery; (4) its mechanism does not depend on host genome integration, thus its effect is transient, reducing off-target effects, limiting toxicity, and improving safety; (5) it has no limitation on coding and delivery capacity (i.e., it simultaneously expresses and delivers all CRISPR/Cas9 components in one dose); (6) it is inexpensive to produce quickly and in large quantities; (7) as an all-in-one system, it eliminates additional manufacturing steps; (8) it can produce and deliver the components of any gene editing system (e.g., TALENs, base editors, and meganucleases) due to its coding versatility and large coding capacity. These innovative features of the SiCRISP delivery platform will help to realize the full potential of gene editing for the treatment of human disease.