Directed evolution of TALE base editors

点击翻译按钮获取中文摘要

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.