提高准确性和安全性的肽插入碱基编辑器工程

IF 12.1 2区 材料科学 Q1 CHEMISTRY, MULTIDISCIPLINARY
Small Pub Date : 2025-02-25 DOI:10.1002/smll.202411583
Qi Chen, Yangning Sun, Jia Yao, Yingfan Lu, Ruikang Qiu, Fuling Zhou, Zixin Deng, Yuhui Sun
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引用次数: 0

摘要

碱基编辑器是引入碱基转换而不发生双链断裂的有效工具,在生物技术和临床领域有着广泛的应用。然而,它们不可忽视的旁观者突变和脱靶效应引起了广泛的安全担忧。为了解决这些问题,开发了一种新的方法,通过将特定的肽片段插入碱基编辑器中脱氨酶的底物结合口袋来修改这些结果。验证了插入肽的组成和位置可以显著影响基于a3的胞嘧啶碱基编辑器和基于tada -8的腺嘌呤碱基编辑器的性能,从而提高人HEK293T细胞的编辑活性和精度。重要的是,在S116残基后插入DPLVLRRRQ肽的tata -8e变体显示出强烈的Y4A5N6基序偏好,在DNA和rna水平上可以准确地编辑目标原间隔中的A5碱基,最小化旁观者效应和脱靶效应。通过总结工程过程中的规律,建立了一套系统的程序,可用于其他类型的碱基编辑器的修改,使其更加准确和安全。此外,肽插入策略也被证明与已有报道的传统氨基酸变化兼容,表现出良好的相容性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Engineering of Peptide-Inserted Base Editors with Enhanced Accuracy and Security

Engineering of Peptide-Inserted Base Editors with Enhanced Accuracy and Security

Engineering of Peptide-Inserted Base Editors with Enhanced Accuracy and Security

Base editors are effective tools for introducing base conversions without double-strand breaks, showing broad applications in biotechnological and clinical areas. However, their non-negligible bystander mutations and off-target effects have raised extensive safety concerns. To address these issues, a novel method is developed by inserting specific peptide fragments into the substrate binding pocket of deaminases in base editors to modify these outcomes. It is validated that the composition and position of the inserted peptide can significantly impact the performance of A3A-based cytosine base editor and TadA-8e-based adenine base editor, leading to improved editing activity and precision in human HEK293T cells. Importantly, the TadA-8e variant with DPLVLRRRQ peptide inserted behind S116 residue showed a strong motif preference of Y4A5N6, which can accurately edit the A5 base in targeted protospacer with minimized bystander and off-target effects in DNA and RNA-level. By summarizing the regularity during engineering, a set of systematic procedures is established, which can potentially be used to modify other types of base editors and make them more accurate and secure. In addition, the peptide insertion strategy is also proven to be compatible with traditional amino acid changes which have been reported, exhibiting excellent compatibility.

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来源期刊
Small
Small 工程技术-材料科学:综合
CiteScore
17.70
自引率
3.80%
发文量
1830
审稿时长
2.1 months
期刊介绍: Small serves as an exceptional platform for both experimental and theoretical studies in fundamental and applied interdisciplinary research at the nano- and microscale. The journal offers a compelling mix of peer-reviewed Research Articles, Reviews, Perspectives, and Comments. With a remarkable 2022 Journal Impact Factor of 13.3 (Journal Citation Reports from Clarivate Analytics, 2023), Small remains among the top multidisciplinary journals, covering a wide range of topics at the interface of materials science, chemistry, physics, engineering, medicine, and biology. Small's readership includes biochemists, biologists, biomedical scientists, chemists, engineers, information technologists, materials scientists, physicists, and theoreticians alike.
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