Efficient multitool/multiplex gene engineering with TALE-BE

TALE base editors are a recent addition to the genome editing toolbox. These molecular tools are fusions of a transcription activator-like effector domain (TALE), split-DddA deaminase halves, and an uracil glycosylase inhibitor (UGI) that have the distinct ability to directly edit double strand DNA, converting a cytosine (C) to a thymine (T). To dissect the editing rules of TALE-BE, we combined the screening of dozens of TALE-BE targeting nuclear genomic loci with a medium/high throughput strategy based on precise knock-in of TALE-BE target site collections into the cell genome. This latter approach allowed us to gain in depth insight of the editing rules in cellulo, while excluding confounding factors such as epigenetic and microenvironmental differences among different genomic loci. Using the knowledge gained, we designed TALE-BE targeting CD52 and achieved very high frequency of gene knock-out (up to 80% of phenotypic CD52 knock out). We further demonstrated that TALE-BE generate only insignificant levels of Indels and byproducts. Finally, we combined two molecular tools, a TALE-BE and a TALEN, for multiplex genome engineering, generating high levels of double gene knock-out (∼75%) without creation of translocations between the two targeted sites.