Leveraging Programmable Integrases for Human Genome Engineering

项目摘要 利用可编程集成技术进行人类基因组工程 基因工程工具箱包括用于基因组操作的各种分子机器， DNA插入方法对生物医学研究的影响最大。基因敲入是 在临床上用于治疗遗传疾病和癌症，在工业上用于生产生物制剂，在农业上用于 改良农作物，研究人类疾病模型，以及其他许多用途。这些 应用通常依赖于由病毒和转座酶介导的随机整合，或位点- 通过同源重组和基因编辑介导的特异性整合。前一类显示高 效率高，但特异性小，而后一类本质上是精确的，但依赖于细胞因子， 因此无效。直到最近才发现了一种新的分子功能， 也是高度准确的：由CRISPR RNA指导的可编程整合酶。 CRISPR系统在过去十年中彻底改变了生物学，因为它可以轻松编程 CRISPR相关核酸酶与引导RNA一起引入DNA双链断裂，即DNA的前体 修复.由于无法轻易地重新设计工程化核酸酶，基因编辑技术一度陷入停滞， RNA引导的DNA靶向的发现消除了这一关键瓶颈。类似的工程瓶颈 整合酶现在可以消除了。 我的中心愿景是开发可编程的RNA引导的整合酶作为一个强大的新平台 人类基因组工程技术。基于我们最近破译序列决定子的工作 这项技术在细菌中的应用，以及扩展CRISPR-Cas亚型的平行研究， 在哺乳动物细胞中，我们将开始系统地努力建立使用这些细胞的能力， 真核细胞中的多亚基整合酶。然后，我们将开发第一个工具， 使用向导RNA文库在数千个不同的基因组靶位点上多重DNA插入事件。 这种方法将使我们能够探索关于非编码元件的作用的基本问题，例如 增强子和绝缘子调节基因表达。此外，我们将利用正交积分来 执行高度程序化的易位事件，并研究复杂基因组重排在 疾病和癌症。我们的研究将为基因工程工具箱提供强大的新工具， 基因组操作的大门，这是任何其他实验方法都无法达到的。

PROJECT SUMMARY LEVERAGING PROGRAMMABLE INTEGRASES FOR HUMAN GENOME ENGINEERING The genetic engineering toolbox comprises a diverse array of molecular machineries for genome manipulation, and DNA insertion methods have arguably had the largest impact on biomedical research. Gene knock-ins are used in the clinic to treat genetic diseases and cancer, in industry to manufacture biologics, in agriculture to improve crops, and in research to generate models of human disease, among many other uses. These applications generally depend on either random integration mediated by viruses and transposases, or site- specific integration mediated by homologous recombination and gene editing. The former category exhibits high efficiency but little specificity, whereas the latter category is inherently precise but reliant on cellular factors and thus ineffective. Only recently has a new molecular functionality been discovered that is both fully autonomous and also highly accurate: programmable integrases directed by CRISPR RNAs. CRISPR systems have revolutionized biology over the past decade because of how easily one can program CRISPR-associated nucleases with guide RNAs to introduce DNA double-strand breaks, the precursor to DNA repair. Whereas the inability to easily redesign engineered nucleases previously stalled gene-editing technology, the discovery of RNA-guided DNA targeting eliminated this critical bottleneck. A similar bottleneck for engineered integrases is now ready for elimination. My central vision is to develop programmable, RNA-guided integrases as a powerful new platform technology for human genome engineering. Building on our recent work that deciphered sequence determinants of this technology in bacteria, as well as parallel studies that expanded the CRISPR–Cas subtypes that function robustly in mammalian cells, we will embark upon a systematic effort to build the capabilities for employing these multi-subunit integrases in eukaryotic cells. We will then develop the first tools for performing simultaneous, multiplexed DNA insertion events across thousands of distinct genomic target sites using guide RNA libraries. This approach will enable us to probe fundamental questions regarding the role of noncoding elements such as enhancers and insulators in regulating gene expression. Furthermore, we will harness orthogonal integrases to execute highly programmed translocation events and study the role of complex genome rearrangements in disease and cancer. Our studies will contribute powerful new tools to the genetic engineering toolbox and open the door to genomic manipulations that are inaccessible with any other experimental approach.