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

小型企业创新研究(SBIR)第一阶段项目的更广泛影响是推动将遗传信息(遗传有效载荷)人工整合到动物细胞、微生物和植物的基因组中。一种新型的分子载体(DNA供体)与已建立的用于基因编辑和基因组工程的CRISPR(簇状短间隔回文重复)系统相结合，可以实现前所未有的基因有效载荷。新的有效载荷交付技术预计将使许多新兴和创新的应用成为可能。这项技术以较低的成本为最先进的方法提供了一种更安全的替代方案。非治疗性应用将使高效地产生细胞系和转基因动物成为可能。在植物生物技术方面，新的DNA捐赠者将只需一轮基因组修改就可以进行复杂的基因工程，提高作物产量和农业生产。拟议的项目为DNA捐赠者提供了一项关键创新，这些DNA捐赠者用于转移基因有效载荷，通过CRISPR进行靶向整合。虽然CRISPR介导的基因破坏(敲除)是非常有效的，但用于靶向整合(敲入)的转基因DNA供体的整合通常是有限的。现有的商业解决方案专注于小基因大小的有效载荷，但迫切需要一种创新的解决方案，能够集成携带几个基因或整个基因电路的更大有效载荷。这项提案中的工作将展示基因转移和编辑技术在治疗相关细胞类型的子集中的广泛适用性，例如干细胞和初级免疫细胞。新型供体载体的性能将通过比较它们与传统DNA供体分子和基于病毒的递送系统的整合效率和整合精度以及细胞毒性来评估。此外，该项目将展示生产大量新DNA捐赠者的能力，从而极大地促进他们在细胞疗法中的应用，并显著降低他们的总体成本。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.