Directed evolution of TALE base editors

该项目旨在进一步开发下一代基因组编辑工具。TALE碱基编辑器可以在活细胞的DNA中引入精确的变化。与基于CRISPR的工具相比，它们的优势在于，它们还可以编辑线粒体和叶绿体的基因组，从而为治疗可遗传的人类疾病和开发具有上级特性的生物工程植物开辟了许多新的机会。此外，与基于CRISPR的工具相比，它们可以更自由地定位以编辑任何所需的目标区域。首先，我们将使用不同的架构来表征和优化现有的TALE碱基编辑器，以实现仅对预期的靶DNA碱基的精确改变。在第二步中，我们将使用替代酶开发新的碱基编辑器。目前的TALE碱基编辑器不能编辑所有的靶胞嘧啶碱基，因为它们具有扩展的靶特异性。因此，我们的目标是开发具有更广泛靶特异性的碱基编辑器，以缓解目前对这些基因组编辑工具应用的限制。为此，将对具有最佳序列特异性但优选作用于单链DNA的替代酶进行定向进化选择筛选，以进化也使用双链DNA的变体。这种进化的酶将帮助我们了解这些蛋白质如何选择它们的靶底物。它将进一步使得能够开发更通用的基于TALE和CRISPR的编辑器，并支持未来具有上级特性的酶的合理设计。最后，我们将在细菌中应用TALE碱基编辑器来表征它们的效率和可能的脱靶活性。在这种方法中，我们将同时在水稻病原细菌的多个毒力基因。这将使我们能够在未来研究这些基因在水稻感染过程中的功能，并支持抗性作物的开发。

This project aims at further developing next generation genome editing tools. TALE base editors can introduce precise changes in the DNA of living cells. Their advantage over CRISPR-based tools is, that they can also edit the genomes of mitochondria and chloroplasts, thereby opening many new chances to treat heritable human diseases and develop bioengineered plants with superior properties. In addition, they can be positioned much more freely in comparison to CRISPR-based tools to edit any desired target region. First, we will characterize and optimize existing TALE base editors using different architectures to enable precise changes of only the intended target DNA base. In a second step, we will develop novel base editors using alternative enzymes. The current TALE base editors can not edit all target cytosine bases, because they have an extended target specificity. Therefore, we aim to develop base editors with a broader target specificity to relieve current restrictions to the application of these genome editing tools. For this, an alternative enzyme which has an optimal sequence specificity, but preferrably acts on single-stranded DNA, will be subjected to a directed evolution selection screen to evolve variants that also use double-stranded DNA. Such an evolved enzyme will help us to understand, how these proteins choose their target substrate. It will further enable the development of more versatile TALE- and CRISPR-base editors and support a future rational design of enzymes with superior properties. Finally, we will apply TALE base editors in bacteria to characterize their efficiency and possible off-target activity. In this approach we will simultaneously inactivate multiple virulence genes in rice-pathogenic bacteria. This will enable us in the future to study the function of these genes during the infection of rice and support the development of resistant crop plants.