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

verfasst von

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.

Organisationseinheit(en)

Institut für Pflanzengenetik

Typ

Artikel

Journal

BMC Biology

Band

Seiten

Anzahl der Seiten

ISSN

1741-7007

Publikationsdatum

29.04.2024

Publikationsstatus

Veröffentlicht

Peer-reviewed

ASJC Scopus Sachgebiete

Allgemeine Agrar- und Biowissenschaften, Ökologie, Evolution, Verhaltenswissenschaften und Systematik, Allgemeine Biochemie, Genetik und Molekularbiologie, Strukturelle Biologie, Physiologie, Biotechnologie, Pflanzenkunde, Zellbiologie, Entwicklungsbiologie

Elektronische Version(en)

https://doi.org/10.1186/s12915-024-01895-0 (Zugang: Offen)