Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA.
Cell
; 187(1): 95-109.e26, 2024 01 04.
Article
in En
| MEDLINE
| ID: mdl-38181745
ABSTRACT
DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA, Mitochondrial
/
Transcription Activator-Like Effectors
Limits:
Animals
/
Humans
Language:
En
Journal:
Cell
Year:
2024
Type:
Article