Details
| Original language | English |
|---|---|
| Article number | 99 |
| Pages (from-to) | 99 |
| Number of pages | 1 |
| Journal | BMC Biology |
| Volume | 22 |
| Issue number | 1 |
| Publication status | Published - 29 Apr 2024 |
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.
Keywords
- Gene Editing/methods, Nicotiana/genetics, Transcription Activator-Like Effectors/metabolism, Cytidine Deaminase/metabolism, Cytosine/metabolism, Oryza/genetics, Base editors, Deaminase, Genome editing, DddA, Chloroplast, Rice
ASJC Scopus subject areas
- Agricultural and Biological Sciences(all)
- Agricultural and Biological Sciences(all)
- Ecology, Evolution, Behavior and Systematics
- Biochemistry, Genetics and Molecular Biology(all)
- Biochemistry, Genetics and Molecular Biology(all)
- Structural Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Physiology
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Agricultural and Biological Sciences(all)
- Plant Science
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Developmental Biology
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In: BMC Biology, Vol. 22, No. 1, 99, 29.04.2024, p. 99.
Research output: Contribution to journal › Article › Research › peer review
}
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 -