Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs.

IF 3.9 3区 医学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Human gene therapy Pub Date : 2024-10-01 Epub Date: 2024-09-24 DOI:10.1089/hum.2024.073
Santiago R Castillo, Brandon W Simone, Karl J Clark, Patricia Devaux, Stephen C Ekker
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引用次数: 0

Abstract

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5' bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5'-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5' bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.

利用 αDdCBEs 进行无约束精准线粒体基因组编辑。
DddA 衍生胞嘧啶碱基编辑器(DdCBEs)能有针对性地将线粒体 DNA(mtDNA)中的 C-G 转换为 T-A。DdCBEs 成对工作,每个臂由一个类似转录激活剂的效应器 (TALE)、半个分裂的双链 DNA 去氨酶和一个尿嘧啶糖基化酶抑制剂组成。这项开创性技术有助于我们更好地了解涉及 mtDNA 的细胞过程,并为开发由致病性 mtDNA 变体引起的遗传疾病的模型和疗法铺平了道路。尽管如此,鉴于 TALE 蛋白的固有特性,人类 mtDNA 中的一些靶位点仍无法被 DdCBE 和其他基于 TALE 的技术所触及。具体来说,由于 TALE 目标序列上游必须有一个胸腺嘧啶的传统要求(即 5'-T 限制),人类线粒体基因组中超过 150 个位点被认为是 DdCBE 无法访问的。以前有人试图通过开发单体 DdCBE 或利用 DNA 结合域替代 TALE 来规避这一要求,但结果都是特异性不理想,治疗潜力降低。为了挑战和阐明 5'-T 约束在 DdCBE 介导的 mtDNA 编辑中的相关性,并扩大该技术可编辑的基团范围,我们生成了含有 TALE 蛋白的 DdCBE,这些 TALE 蛋白经过工程化处理后可识别所有 5' 碱基。这些经过修饰的 DdCBE 在此称为 αDdCBE。值得注意的是,不符合5'-T规范的DdCBE能有效编辑不同位点的mtDNA。然而,αDdCBEs 的表现却常常优于它们,无论它们的 TALE 结合位点的 5' 碱基是多少,αDdCBEs 的活性和特异性都有显著提高。此外,我们还发现 αDdCBE 与增强型 DddAtox 变体 DddA6 和 DddA11 兼容,并验证了 TALE 与 αDdCBE 的转移是优化碱基编辑结果的有效方法。总之,αDdCBEs 可以实现高效、特异和无约束的线粒体碱基编辑。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Human gene therapy
Human gene therapy 医学-生物工程与应用微生物
CiteScore
6.50
自引率
4.80%
发文量
131
审稿时长
4-8 weeks
期刊介绍: Human Gene Therapy is the premier, multidisciplinary journal covering all aspects of gene therapy. The Journal publishes in-depth coverage of DNA, RNA, and cell therapies by delivering the latest breakthroughs in research and technologies. Human Gene Therapy provides a central forum for scientific and clinical information, including ethical, legal, regulatory, social, and commercial issues, which enables the advancement and progress of therapeutic procedures leading to improved patient outcomes, and ultimately, to curing diseases.
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