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Today we report in @nature, in collaboration with the labs of Joseph Mougous and @VamsiMootha, the development of a new class of CRISPR-free base editors that enable precision editing of mitochondrial DNA (mtDNA) for the first time. drive.google.com/file/d/1xHcs__… 1/7
The team discovered DddA, an interbacterial toxin that is a novel cytidine deaminase enzyme that operates on *double-stranded* DNA (dsDNA), unlike all previously known cytidine deaminases, which require single-stranded DNA (ssDNA). 2/7
Previous efforts to precisely edit mtDNA have been stymied by the fact that CRISPR requires guide RNAs, which have not been effectively delivered into the mitochondria. mtDNA can be cut with ZFNs or TALENs, but doing so results in mtDNA destruction rather than precise editing.3/7
Since DddA can deaminate dsDNA, we envisioned that it could be used to create a new class of base editors that did not require CRISPR or guide RNAs to create ssDNA regions, which are normally targeted by standard CRISPR base editors. 4/7
Indeed, splitting DddA into two non-toxic halves and fusing each half to a TALE array programmed to bind adjacent DNA target sites resulted in DdCBE, a novel cytosine base editor that does not use CRISPR and thus can edit mtDNA. 5/7
We used DdCBE to edit 5 mtDNA genes in human cells, converting targeted C•G base pairs to T•A. Editing MT-ND4 changed mitochondrial phenotypes in expected ways. DdCBE editing was reasonably efficient (~10-50%), durable, and proceeded with low levels of off-target editing. 6/7
Congratulations to Beverly, Marcos, Jun, Dustin, Anna, Aditya, FoSheng, Matthew, Brook, Vamsi, and Joseph! SI: drive.google.com/file/d/1S2dZkP… 7/7
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