SBIR Phase I: Development of a safe gene editing system via CRISPR-Cas and Cas inhibitor co-delivery

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The broader impact of this Small Business Innovation Research (SBIR) Phase I project is to enable safe and effective human gene editing, providing therapies targeting myriad human genetic diseases. CRISPR (clustered, regularly interspaced, short palindromic repeats) and CRISPR-associated (Cas) genes compose adaptive microbial `immune systems' found in diverse bacterial species, serving as a defense mechanism against viral infection. The simplicity, programmability, and versatility of CRISPR-Cas systems have enabled genetic modification of many organisms and offer immense therapeutic potential for treating human diseases. However, CRISPR-based gene editing can also cause off-target edits, resulting in the introduction of mutations, insertions, deletions, or DNA restructuring at unintended off-target editing. This effect can cause significant problems. The proposed technology will develop tools to safely translate CRISPR-based gene editing to in vivo human therapeutics. This Small Business Innovation Research (SBIR) Phase I project is to advance a technology based on virus-encoded CRISPR-Cas inhibitor off-switches, enabling control of off-target gene editing. These anti-CRISPR proteins are a novel class of robust protein inhibitors that can be genetically encoded for co-delivery with the CRISPR editing machinery. This proposal will focus on understanding CRISPR editing kinetics to pinpoint the ideal ‘editing window’ providing the highest therapeutic benefit while minimizing off-target risk. Second, the project will design a system for simultaneous delivery of the CRISPR editing machinery along with the anti-CRISPR inhibitor to leverage this new ‘editing window’. Genetic regulatory elements will be used to tune the expression of the various components of the system and provide the framework for a therapeutic delivery system. This work will enable safe and effective gene editing in a therapeutically relevant context, providing a toolkit for translation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.