加速发现新型单链胞苷脱氨酶并使其微型化

IF 16.6 2区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nucleic Acids Research Pub Date : 2024-09-14 DOI:10.1093/nar/gkae800

Jiacheng Deng, Xueyuan Li, Hao Yu, Lin Yang, Ziru Wang, Wenfeng Yi, Ying Liu, Wenyu Xiao, Hongyong Xiang, Zicong Xie, Dongmei Lv, Hongsheng Ouyang, Daxin Pang, Hongming Yuan

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引用次数: 0

摘要

胞嘧啶碱基编辑器（CBEs）在遗传疾病治疗和动物优良性状培育方面具有巨大潜力。然而，由于腺相关病毒（AAV）的包装容量为 4.7 kb，其巨大的蛋白质尺寸限制了其通过腺相关病毒（AAV）的传输。为了克服这一问题，我们采用了基于网络的快速通用发现（WFG）策略，确定了几种小型 ssDNA 去氨酶（Sdds），并构建了多个 Sdd-CBE 1.0 版本。SflSdd-CBE 1.0 表现出很高的 C-T 编辑效率，与 AncBE4max 相当，而 SviSdd-CBE 1.0 表现出中等的 C-T 编辑效率，编辑窗口较窄（C3 到 C5）。利用 AlphaFold2，我们设计了一种一步到位的微型化策略，在保持其效率的同时缩小了 Sdds 的尺寸。值得注意的是，我们给小鼠注射了表达 PCSK9 靶向 sgRNA 和 SflSdd-CBEs （nSaCas9）2.0 的 AAV8，导致了基因编辑事件（编辑效率高达 15%）和血清胆固醇水平的降低，突出了 Sdds 在基因治疗中的潜力。这些发现为治疗罕见遗传病提供了新的单链编辑工具。

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Accelerated discovery and miniaturization of novel single-stranded cytidine deaminases

Cytidine base editors (CBEs) hold significant potential in genetic disease treatment and in breeding superior traits into animals. However, their large protein sizes limit their delivery by adeno-associated virus (AAV), given its packing capacity of <4.7 kb. To overcome this, we employed a web-based fast generic discovery (WFG) strategy, identifying several small ssDNA deaminases (Sdds) and constructing multiple Sdd-CBE 1.0 versions. SflSdd-CBE 1.0 demonstrated high C-to-T editing efficiency, comparable to AncBE4max, while SviSdd-CBE 1.0 exhibited moderate C-to-T editing efficiency with a narrow editing window (C3 to C5). Utilizing AlphaFold2, we devised a one-step miniaturization strategy, reducing the size of Sdds while preserving their efficiency. Notably, we administered AAV8 expressing PCSK9 targeted sgRNA and SflSdd-CBEs (nSaCas9) 2.0 into mice, leading to gene-editing events (with editing efficiency up to 15%) and reduced serum cholesterol levels, underscoring the potential of Sdds in gene therapy. These findings offer new single-stranded editing tools for the treatment of rare genetic diseases.

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来源期刊

Nucleic Acids Research 生物-生化与分子生物学

CiteScore

27.10

自引率

4.70%

发文量

1057

审稿时长

2 months

期刊介绍： Nucleic Acids Research (NAR) is a scientific journal that publishes research on various aspects of nucleic acids and proteins involved in nucleic acid metabolism and interactions. It covers areas such as chemistry and synthetic biology, computational biology, gene regulation, chromatin and epigenetics, genome integrity, repair and replication, genomics, molecular biology, nucleic acid enzymes, RNA, and structural biology. The journal also includes a Survey and Summary section for brief reviews. Additionally, each year, the first issue is dedicated to biological databases, and an issue in July focuses on web-based software resources for the biological community. Nucleic Acids Research is indexed by several services including Abstracts on Hygiene and Communicable Diseases, Animal Breeding Abstracts, Agricultural Engineering Abstracts, Agbiotech News and Information, BIOSIS Previews, CAB Abstracts, and EMBASE.