CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA
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TLDR
In this article , the authors used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope, which substantially increase the effectiveness and applicability of all-protein base editing.Abstract:
The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C•G-to-T•A editing. To improve editing efficiency and overcome the strict TC sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at TC by 3.3-fold on average. DdCBEs containing evolved DddA11 offered a broadened HC (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at AC and CC targets from less than 10% for canonical DdCBE to 15-30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-TC target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing. read more
Citations
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Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity
Monica E. Neugebauer,Alvin Hsu,Mandana Arbab,Nicholas A Krasnow,Amber N. McElroy,Smriti Pandey,Jordan L. Doman,Tony P. Huang,Aditya Raguram,Samagya Banskota,Gregory A. Newby,Jakub Tolar,Mark J. Osborn,David R. Liu +13 more
TL;DR: In this paper , the TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity.
Journal ArticleDOI
Mitochondrial genome engineering coming-of-age.
TL;DR: Barrera-Paez et al. as discussed by the authors used a base editor with DNA double-strand deoxyadenosine deaminase to modify the mtDNA in vitro.
Journal ArticleDOI
Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors
TL;DR: HiFi-DdCBE as discussed by the authors is a high-fidelity DddA-derived cytosine base editors with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DDDA tox halves.
Journal ArticleDOI
DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing
Li Mi,Ming Shi,Yu Xuan Li,Gang Xie,Xichen Rao,Damu Wu,Aimin Cheng,Mengxiao Niu,Fengli Xu,Ying Yu,Ning Gao,Wensheng Wei,Xianhua Wang,Yangming Wang +13 more
TL;DR: In this paper , the authors identify a homolog from Simiaoa sunii (Ddd_Ss) which can efficiently deaminate cytosine in D C context in double-stranded DNA (dsDNA).
Journal ArticleDOI
Mitochondrial DNA is a major source of driver mutations in cancer.
TL;DR: The role of mutations in the mtDNA genomes of tumors has been contentious as discussed by the authors , and a conceptual fog surrounding the functional impact of mtDNA mutations in tumors has begun to lift, revealing a path to understanding the role of this essential metabolic genome in cancer initiation and progression.
References
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SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA/Q File Manipulation.
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