EditR: A Method to Quantify Base Editing from Sanger Sequencing
EditR: A Method to Quantify Base Editing from Sanger Sequencing
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DOI：
10.1089/crispr.2018.0014
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发表时间：
2018-06-01
期刊：
影响因子：
3.7
通讯作者：
Moriarity, Branden S.
中科院分区：
文献类型：
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作者：
Kluesner, Mitchell G.;Nedveck, Derek A.;Moriarity, Branden S.
CRISPR-Cas9-胞苷脱氨酶融合酶被称为碱基编辑器，允许基因组脱氧胞苷到脱氧胸苷（C：GT：A）的低靶向编辑，而不需要双链断裂诱导。碱基编辑器代表了基因编辑技术的范式转变，因为它们具有前所未有的介导靶向单碱基转换的效率。然而，目前对碱基编辑结果的分析依赖于不精确或昂贵且耗时的方法。为了克服这些局限性，我们开发了一种简单、成本效益高且准确的程序来测量基于荧光的桑格测序的碱基编辑效率，称为EditR。我们提供EditR作为一个免费的在线工具或可下载的桌面应用程序，需要一个单一的桑格测序文件和指导RNA序列。EditR比酶促测定更准确，并提供了对碱基编辑的位置、类型和效率的额外见解。此外，EditR可能适合于从最近开发的作用于DNA（腺苷脱氨酶碱基编辑器[ABE]）或RNA（REPAIR）（催化A：TG：C）的腺苷脱氨酶碱基编辑器中量化碱基编辑。总的来说，我们证明了EditR是一个强大的，廉价的工具，将促进基础编辑技术的广泛应用，从而促进在这个新兴领域的进一步创新。
CRISPR-Cas9-Cytidine deaminase fusion enzymestermed base editorsallow targeted editing of genomic deoxycytidine to deoxythymidine (C:GT:A) without the need for double-stranded break induction. Base editors represent a paradigm shift in gene editing technology due to their unprecedented efficiency to mediate targeted, single-base conversion. However, current analysis of base editing outcomes rely on methods that are either imprecise or expensive and time-consuming. To overcome these limitations, we developed a simple, cost-effective, and accurate program to measure base editing efficiency from fluorescence-based Sanger sequencing, termed EditR. We provide EditR as a free online tool or downloadable desktop application requiring a single Sanger sequencing file and guide RNA sequence. EditR is more accurate than enzymatic assays, and provides added insight to the position, type, and efficiency of base editing. Furthermore, EditR is likely amenable to quantify base editing from the recently developed adenosine deaminase base editors that act on either DNA (adenosine deaminase base editors [ABEs]) or RNA (REPAIRs) (catalyzes A:TG:C). Collectively, we demonstrate that EditR is a robust, inexpensive tool that will facilitate the broad application of base editing technology, thereby fostering further innovation in this burgeoning field.