SBIR Phase I: A novel class of molecular vehicles for the targeted and precise integration of specified genetic information into the genomes of host cells and organisms

The broader impact of this Small Business Innovation Research (SBIR) Phase I project is to advance artificial integration of genetic information (genetic payloads) into the genomes of animal cells, microorganisms, and plants. A novel class of molecular vehicles (DNA donors) allows an unprecedented efficiency in delivering genetic payloads when combined with the established CRISPR (clustered short interspaced palindromic repeats) system for gene editing and genome engineering. The new payload delivery technology is expected to enable many emergent and innovative applications. This technology provides a safer alternative to the state-of-the-art method at lower cost. Non-therapeutic applications will enable the efficient generation of cell lines and transgenic animals. In plant biotechnology, the new DNA donors will allow complex genetic engineering with only one round of genome modification, improving crop yields and agricultural production. The proposed project provides a key innovation to the DNA donors that are used for transferring genetic payloads for targeted integration via CRISPR. Whereas CRISPR-mediated gene disruption (knock-out) is very efficient, integration of transfected DNA donors for targeted integration (knock-in) is generally limited. Existing commercial solutions are focused on small gene-sized payloads, but an innovative solution that enables the integration of larger payloads carrying several genes or entire genetic circuits is critically needed. The work in this proposal will demonstrate the broad applicability of the gene transfer and editing technology in a subset of therapeutically relevant cell types, such as stem cells and primary immune cells. The performance of the novel donor vehicles will be assessed by comparing their integration efficiency and precision of integration and cell toxicity to conventional DNA donor molecules and virus-based delivery systems. In addition, the project will demonstrate the ability to produce large quantities of the new DNA donors, thus greatly facilitating their application in cell therapies and dramatically reducing their overall cost. 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.