Kluesner Mitchell G, Nedveck Derek A, Lahr Walker S, Garbe John R, Abrahante Juan E, Webber Beau R, Moriarity Branden S
1 Masonic Cancer Center, University of Minnesota , Minneapolis, Minnesota.
2 Department of Pediatrics, University of Minnesota , Minneapolis, Minnesota.
CRISPR J. 2018 Jun;1(3):239-250. doi: 10.1089/crispr.2018.0014.
CRISPR-Cas9-Cytidine deaminase fusion enzymes-termed "base editors"-allow targeted editing of genomic deoxycytidine to deoxythymidine (C:G→T: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:T→G: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.
CRISPR-Cas9-胞苷脱氨酶融合酶(称为“碱基编辑器”)可将基因组脱氧胞苷定向编辑为脱氧胸苷(C:G→T:A),而无需诱导双链断裂。碱基编辑器代表了基因编辑技术的范式转变,因为它们介导定向单碱基转换的效率前所未有的高。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。为克服这些局限性,我们开发了一个简单、经济高效且准确的程序,用于从基于荧光的桑格测序中测量碱基编辑效率,称为“EditR”。我们将EditR作为免费在线工具或可下载的桌面应用程序提供,只需一个桑格测序文件和引导RNA序列。EditR比酶促测定更准确,并能提供有关碱基编辑位置、类型和效率的更多信息。此外,EditR可能适用于量化最近开发的作用于DNA(腺苷脱氨酶碱基编辑器[ABEs])或RNA(REPAIRs)(催化A:T→G:C)的腺苷脱氨酶碱基编辑器的碱基编辑。总体而言,我们证明EditR是一种强大、廉价的工具,将促进碱基编辑技术的广泛应用,从而推动这一新兴领域的进一步创新。