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

机译：DNA碱基编辑诱导的偏离靶RNA突变及其消除

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摘要

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks(1-3), 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(4-8), it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA(9-13). For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA(12), and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA(9,11). 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(14-16). 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.
机译：最近开发的DNA碱基编辑方法使得基因组DNA中的直接产生所需的点突变，而不产生任何双链断裂（1-3），但偏离目标编辑的问题限制了这些方法的应用。尽管以前的几种研究已经评估了基因组DNA（4-8）中的脱靶突变，但现在清楚的是，与常用的DNA碱基编辑器是一体的脱氨酶通常与RNA（9-13）结合。例如，在胞嘧啶基础编辑器（CBE）中用于胞嘧啶脱氨酶Apobec1- -Targets DNA和RNA（12），以及用于腺嘌呤基础编辑器（ABES）的腺嘌呤脱氨酶TADA - 抑制特异性杀虫素形成RNA（9,11）。然而，尚未评估由DNA碱基编辑引起的任何潜在的RNA突变。腺相关病毒是涉及DNA编辑的基因疗法最常见的输送系统;这些病毒可以在体内维持长期基因表达，因此DNA碱基编辑诱导的潜在RNA突变的程度非常关注（14-16）。在这里，我们定量评估了CBE或ABES诱导的RNA单核苷酸变化（SNV）。胞嘧啶基础编辑器Be3和Adenine Base Editor Abe7.10都产生了数万个偏离目标RNA SNV。随后，通过工程脱氨酶，我们发现三个CBE变体和一个ABE变体显示出偏离靶RNA SNV的偏离靶RNA SNV，同时保持有效的DNA对靶活性。本研究揭示了DNA编辑中偏离目标效果的先前被忽视的方面，并且还证明了这种效果可以通过工程脱氨酶消除。

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  • 来源
    《Nature》 |2019年第7764期|275-278|共4页
  • 作者

    Zhou Changyang; Sun Yidi; Yan Rui; Liu Yajing; Zuo Erwei; Gu Chan; Han Linxiao; Wei Yu; Hu Xinde; Zeng Rong; Li Yixue; Zhou Haibo; Guo Fan; Yang Hui;

  • 作者单位

    Chinese Acad Sci Inst Neurosci Key Lab Primate Neurobiol State Key Lab Neurosci CAS Ctr Excellence Brain S Shanghai Inst Biol Sci Shanghai Res Ctr Brain Sci Shanghai Peoples R China|Univ Chinese Acad Sci Coll Life Sci Beijing Peoples R China;

    Univ Chinese Acad Sci Coll Life Sci Beijing Peoples R China|Chinese Acad Sci CAS Key Lab Syst Biol CAS Ctr Excellence Mol Cell Inst Biochem & Cell Biol Shanghai Inst Biol Sci Shanghai Peoples R China|Chinese Acad Sci Univ Chinese Acad Sci Key Lab Computat Biol Biomed Big Data Ctr CAS MPG Partner Inst Computat Shanghai Inst Nutr & Hlth Shanghai Inst Biol Sci Shanghai Peoples R China;

    Sichuan Univ Coll Life Sci West China Univ Hosp 2 Ctr Translat Med Minist Educ Dept Obstet & Gynecol Key Lab Birth Defects & Rel Chengdu Sichuan Peoples R China;

    Univ Chinese Acad Sci Coll Life Sci Beijing Peoples R China|Shanghai Tech Univ Sch Life Sci & Technol Shanghai Peoples R China;

    Chinese Acad Sci Inst Neurosci Key Lab Primate Neurobiol State Key Lab Neurosci CAS Ctr Excellence Brain S Shanghai Inst Biol Sci Shanghai Res Ctr Brain Sci Shanghai Peoples R China|Chinese Acad Agr Sci Agr Genome Inst Shenzhen Ctr Anim Genom Shenzhen Peoples R China;

    Sichuan Univ Coll Life Sci West China Univ Hosp 2 Ctr Translat Med Minist Educ Dept Obstet & Gynecol Key Lab Birth Defects & Rel Chengdu Sichuan Peoples R China;

    Chinese Acad Sci Inst Neurosci Key Lab Primate Neurobiol State Key Lab Neurosci CAS Ctr Excellence Brain S Shanghai Inst Biol Sci Shanghai Res Ctr Brain Sci Shanghai Peoples R China;

    Chinese Acad Sci Inst Neurosci Key Lab Primate Neurobiol State Key Lab Neurosci CAS Ctr Excellence Brain S Shanghai Inst Biol Sci Shanghai Res Ctr Brain Sci Shanghai Peoples R China;

    Chinese Acad Sci Inst Neurosci Key Lab Primate Neurobiol State Key Lab Neurosci CAS Ctr Excellence Brain S Shanghai Inst Biol Sci Shanghai Res Ctr Brain Sci Shanghai Peoples R China|Univ Chinese Acad Sci Coll Life Sci Beijing Peoples R China;

    Chinese Acad Sci CAS Key Lab Syst Biol CAS Ctr Excellence Mol Cell Inst Biochem & Cell Biol Shanghai Inst Biol Sci Shanghai Peoples R China|Shanghai Tech Univ Sch Life Sci & Technol Shanghai Peoples R China;

    Sichuan Univ Coll Life Sci West China Univ Hosp 2 Ctr Translat Med Minist Educ Dept Obstet & Gynecol Key Lab Birth Defects & Rel Chengdu Sichuan Peoples R China|Shanghai Tech Univ Sch Life Sci & Technol Shanghai Peoples R China|Fudan Univ Shanghai Acad Sci & Technol Shanghai Jiao Tong Univ Shanghai Peoples R China;

    Chinese Acad Sci Inst Neurosci Key Lab Primate Neurobiol State Key Lab Neurosci CAS Ctr Excellence Brain S Shanghai Inst Biol Sci Shanghai Res Ctr Brain Sci Shanghai Peoples R China;

    Sichuan Univ Coll Life Sci West China Univ Hosp 2 Ctr Translat Med Minist Educ Dept Obstet & Gynecol Key Lab Birth Defects & Rel Chengdu Sichuan Peoples R China;

    Chinese Acad Sci Inst Neurosci Key Lab Primate Neurobiol State Key Lab Neurosci CAS Ctr Excellence Brain S Shanghai Inst Biol Sci Shanghai Res Ctr Brain Sci Shanghai Peoples R China;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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