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DNA 碱基编辑诱导的脱靶 RNA 突变及其通过诱变的消除。

Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis.

机构信息

Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China.

出版信息

Nature. 2019 Jul;571(7764):275-278. doi: 10.1038/s41586-019-1314-0. Epub 2019 Jun 10.


DOI:10.1038/s41586-019-1314-0
PMID:31181567
Abstract

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA, and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.

摘要

最近开发的 DNA 碱基编辑方法能够在不产生任何双链断裂的情况下直接生成所需的点突变,但脱靶编辑的问题限制了这些方法的应用。尽管之前有几项研究评估了基因组 DNA 中的脱靶突变,但现在很清楚,常用于 DNA 碱基编辑器的脱氨酶通常与 RNA 结合。例如,胞嘧啶脱氨酶 APOBEC1-用于胞嘧啶碱基编辑器 (CBEs)-既靶向 DNA 又靶向 RNA,而腺嘌呤脱氨酶 TadA-用于腺嘌呤碱基编辑器 (ABEs)-在 RNA 上诱导特异性的肌苷形成。然而,尚未评估 DNA 碱基编辑器引起的任何潜在 RNA 突变。腺相关病毒是涉及 DNA 编辑的基因治疗最常用的递送系统;这些病毒可以在体内维持长期的基因表达,因此,DNA 碱基编辑器诱导的潜在 RNA 突变的程度令人非常关注。在这里,我们定量评估了 CBE 或 ABE 诱导的 RNA 单核苷酸变异 (SNV)。胞嘧啶碱基编辑器 BE3 和腺嘌呤碱基编辑器 ABE7.10 都产生了数万种脱靶 RNA SNV。随后,通过工程化脱氨酶,我们发现三种 CBE 变体和一种 ABE 变体在保持高效 DNA 靶标活性的同时,减少了脱靶 RNA SNV 至基线水平。这项研究揭示了 DNA 编辑中脱靶效应的一个以前被忽视的方面,也证明了通过工程化脱氨酶可以消除这种效应。

相似文献

[1]
Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis.

Nature. 2019-6-10

[2]
Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing.

Commun Biol. 2021-7-16

[3]
TadA orthologs enable both cytosine and adenine editing of base editors.

Nat Commun. 2023-1-26

[4]
Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors.

Nature. 2019-4-17

[5]
Sequence-specific prediction of the efficiencies of adenine and cytosine base editors.

Nat Biotechnol. 2020-7-6

[6]
High-Fidelity Cytosine Base Editing in a GC-Rich Corynebacterium glutamicum with Reduced DNA Off-Target Editing Effects.

Microbiol Spectr. 2022-12-21

[7]
Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing.

Nat Biotechnol. 2023-5

[8]
Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity.

Nat Commun. 2021-4-16

[9]
Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity.

Nat Biotechnol. 2023-5

[10]
CRISPR DNA base editors with reduced RNA off-target and self-editing activities.

Nat Biotechnol. 2019-9-2

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An integrated enzymatic and computational pipeline for quantifying off-target base-editing.

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[3]
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Animals (Basel). 2025-7-22

[4]
PAM-flexible adenine base editing rescues hearing loss in a humanized MPZL2 mouse model harboring an East Asian founder mutation.

Nat Commun. 2025-8-5

[5]
CRISPR-based functional genomics tools in vertebrate models.

Exp Mol Med. 2025-7

[6]
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[7]
precision base editing to rescue mouse models of disease.

Mol Ther Nucleic Acids. 2025-7-1

[8]
Advancements in CRISPR/Cas systems for disease treatment.

Acta Pharm Sin B. 2025-6

[9]
Establishment and rescue of fibroblast cell lines carrying a nonsense mutation of RB1 by CRISPR-based base editing.

Sci Rep. 2025-7-11

[10]
High-precision cytosine base editors by evolving nucleic-acid-recognition hotspots in deaminase.

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