Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants

authored by

Dingbo Zhang, Vanessa Pries, Jens Boch

Abstract

BACKGROUND: TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C•G-to-T•A editing. This technology has not been optimized for use in plant cells.

RESULTS: To systematically investigate TALE-DdCBE architectures and editing rules, we established a β-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C•G-to-T•A conversions in their chloroplast genome, as well as N. benthamiana with C•G-to-T•A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants.

CONCLUSIONS: Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement.

Organisation(s)

Institute of Plant Genetics

Type

Article

Journal

BMC Biology

Volume

Pages

No. of pages

ISSN

1741-7007

Publication date

29.04.2024

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Agricultural and Biological Sciences(all), Ecology, Evolution, Behavior and Systematics, Biochemistry, Genetics and Molecular Biology(all), Structural Biology, Physiology, Biotechnology, Plant Science, Cell Biology, Developmental Biology

Electronic version(s)

https://doi.org/10.1186/s12915-024-01895-0 (Access: Open)