CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA.
CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA.
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无CRISPR基础编辑器具有增强的活性，并扩大了线粒体和核DNA的靶向范围。
DOI：
10.1038/s41587-022-01256-8
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发表时间：
2022-09
影响因子：
46.9
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
Mok, Beverly Y.;Kotrys, Anna, V;Raguram, Aditya;Huang, Tony P.;Mootha, Vamsi K.;Liu, David R.
全蛋白胞嘧啶碱基编辑器DdCBE使用TALE蛋白和双链DNA特异性胞苷脱氨酶（DddA）来介导靶向C·G至T·A编辑。为了提高编辑效率和克服DddA严格的TC序列上下文约束，我们使用噬菌体辅助的非连续和连续进化来进化具有改进活性和扩大靶向范围的DddA变体。与典型的DdCBE相比，具有进化的DddA 6的碱基编辑器在TC时将线粒体DNA（mtDNA）编辑效率平均提高了3.3倍。含有进化的DddA 11的DdCBE为线粒体和核碱基编辑提供了更广泛的HC（H = A，C或T）序列兼容性，将AC和CC靶点的平均编辑效率从典型DdCBE的不到10%提高到15-30%，并在分选以表达DdCBE的两个半部分的细胞群中提高到50%。我们使用这些进化的DdCBE在人类细胞中的非TC靶位点有效地安装疾病相关的mtDNA突变。DddA 6和DddA 11大大提高了全蛋白碱基编辑的有效性和适用性。不断进化的双链DNA脱氨酶提高了全蛋白碱基编辑器的效率和靶向范围。
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. Continuously evolved double-stranded DNA deaminases increase the efficiency and targeting scope of all-protein base editors.
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影响因子：
16.6
作者：
Jain S;Shukla S;Yang C;Zhang M;Fatma Z;Lingamaneni M;Abesteh S;Lane ST;Xiong X;Wang Y;Schroeder CM;Selvin PR;Zhao H
通讯作者：
Zhao H
影响因子：
64.8
作者：
Komor AC;Kim YB;Packer MS;Zuris JA;Liu DR
通讯作者：
Liu DR
影响因子：
16.6
作者：
Badran AH;Liu DR
通讯作者：
Liu DR
影响因子：
9.5
作者：
Diroma, Maria Angela;Ciaccia, Loredana;Picardi, Ernesto
通讯作者：
Picardi, Ernesto
影响因子：
16.6
作者：
Nissanka N;Bacman SR;Plastini MJ;Moraes CT
通讯作者：
Moraes CT