TY - JOUR

T1 - Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants

AU - Zhang, Dingbo

AU - Pries, Vanessa

AU - Boch, Jens

N1 - Open Access funding enabled and organized by Projekt DEAL. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG; BO 1496/9–1).

PY - 2024/4/29

Y1 - 2024/4/29

N2 - 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.

AB - 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.

KW - Gene Editing/methods

KW - Nicotiana/genetics

KW - Transcription Activator-Like Effectors/metabolism

KW - Cytidine Deaminase/metabolism

KW - Cytosine/metabolism

KW - Oryza/genetics

KW - Base editors

KW - Deaminase

KW - Genome editing

KW - DddA

KW - Chloroplast

KW - Rice

UR - http://www.scopus.com/inward/record.url?scp=85191738970&partnerID=8YFLogxK

U2 - 10.1186/s12915-024-01895-0

DO - 10.1186/s12915-024-01895-0

M3 - Article

C2 - 38679734

VL - 22

SP - 99

JO - BMC Biology

JF - BMC Biology

SN - 1741-7007

IS - 1

M1 - 99

ER -