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

这个小型企业创新研究(SBIR)第一阶段项目寻求开发一种专门从培养细胞中移除大片基因组DNA的技术。基因组编辑技术通过实现有针对性的基因座敲除或替换，改变了细胞生物学。这些编辑允许在用于基础研究和药物发现应用的细胞和动物模型中引入与疾病相关的突变。目前的技术依赖于定制的酶来切割特定的基因组位置，因此它们并行切割多个基因组位置的能力有限。Cas9是一种新近发现的细菌蛋白，是一种由RNA引导的DNA内切酶。该公司将测试Cas9同时在两个位置切割小鼠基因组DNA的能力，并从基因组中删除中间序列。通过将Cas9分别定位于近距离和远距离基因座，短和长基因组基因座都将被移除。如果该项目成功，该项目的更广泛影响/商业潜力将是能够产生大规模基因组删除和重排引起的遗传病的细胞和动物模型。成功地证明了在培养细胞中切除基因组基因的能力，将使这项技术的发展成为可能，用于切除小鼠胚胎基因组DNA的区域，并产生新的动物疾病模型。大规模的缺失可以移除内含子、外显子、基因间隔区，甚至整个基因簇，重述遗传性疾病的致病突变，而目前可用的基因组工程技术很难满足这一需求。利用这项技术开发的细胞和动物模型将进一步进行基础研究，了解特定遗传位点如何控制生物途径，并将有助于识别和开发新的药物靶点。

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.