SBIR Phase I: Engineered large-scale genomic deletions with a universal targeted cleavage system

这个小企业创新研究（SBIR）第一阶段项目旨在开发一种技术，专门从培养细胞中去除大区域的基因组DNA。基因组编辑技术通过实现基因组基因座的靶向敲除或替换而改变了细胞生物学。这些编辑允许在细胞和动物模型中引入疾病相关突变，用于基础研究和药物发现应用。目前的技术依赖于定制的酶来切割特定的基因组位点，因此限制了它们平行切割多个基因组位点的能力。Cas9是最近发现的细菌蛋白，其是RNA引导的DNA内切核酸酶。该公司将测试Cas9同时在两个位置切割小鼠基因组DNA并从基因组中切除插入序列的能力。通过将Cas9分别靶向到附近和远处的基因座，短基因组和长基因组基因座都将被去除。如果成功，该项目更广泛的影响/商业潜力将是能够产生由大规模基因组缺失和重排引起的遗传疾病的细胞和动物模型。切除培养细胞中基因组位点的能力的成功证明将允许开发用于切除小鼠胚胎基因组DNA区域的该技术和产生新的动物疾病模型。大规模缺失可以去除内含子、外显子、基因间区域或甚至整个基因簇，重现遗传性疾病的致病突变，这是目前可用的基因组工程技术难以满足的需求。使用该技术开发的细胞和动物模型将进一步促进基础研究和对特定遗传位点如何控制生物途径的理解，并将促进新药物靶点的鉴定和开发。

This Small Business Innovation Research (SBIR) Phase I project seeks to develop a technology for specifically removing large regions of genomic DNA from culture cells. Genome editing technologies have transformed cell biology by enabling the targeted knock-out or replacement of genomic loci. These edits permit the introduction of disease-relevant mutations in cellular and animal models for basic research and drug discovery applications. Current technologies rely on custom enzymes to cleave specific genomic sites, and are therefore limited in their ability to cleave multiple genomic sites in parallel. Cas9 is a recently discovered bacterial protein that is an RNA-guided DNA endonuclease. The company will test the ability of Cas9 to cleave mouse genomic DNA in two locations simultaneously and excise the intervening sequence from the genome. Both short and long genomic loci will be removed by targeting Cas9 to nearby and distant loci, respectively.The broader impact/commercial potential of this project, if successful, will be the ability to generate cellular and animal models of genetic diseases caused by large-scale genomic deletions and rearrangements. Successful demonstration of the ability to excise genomic loci in cultured cells will permit the development of this technology for excising regions of mouse embryo genomic DNA and the generation of new animal disease models. Large scale deletions can remove introns, exons, intergenic regions, or even entire gene clusters, recapitulating causative mutations of inherited disorders, a need poorly met by currently available genome engineering technologies. The cellular and animal models developed using this technology will further basic research and the understanding of how particular genetic loci control biological pathways and will facilitate the identification and development of new drug targets.