Division of Immune Diversity, Program in Cancer Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Faculty of Biosciences, Heidelberg University, Heidelberg, Germany.
Methods Mol Biol. 2021;2181:51-67. doi: 10.1007/978-1-0716-0787-9_4.
The AID/APOBEC family of enzymes are cytidine deaminases that act upon DNA and RNA. Among APOBECs, the best characterized family member to act on RNA is the enzyme APOBEC1. APOBEC1-mediated RNA editing plays a key role in lipid metabolism and in maintenance of brain homeostasis. Editing can be easily detected in RNA-seq data as a cytosine to thymine (C-to-T) change with regard to the reference. However, there are many other sources of base conversions relative to reference, such as PCR errors, SNPs, and even DNA editing by mutator APOBECs. Furthermore, APOBEC1 exhibits disparate activity in different cell types, with respect to which transcripts are edited and the level to which they are edited. When considering these potential sources of error and variability, an RNA-seq comparison between wild-type APOBEC1 sample and a matched control with an APOBEC1 knockout is a reliable method for the discrimination of true sites edited by APOBEC1. Here we present a detailed description of a method for studying APOBEC1 RNA editing, specifically in the murine macrophage cell line RAW 264.7. Our method covers the production of an APOBEC1 knockout cell line using the CRISPR/Cas9 system, through to experimental validation and quantification of editing sites (where we discuss a recently published algorithm (termed MultiEditR) which allows for the detection and quantification of RNA editing from Sanger sequencing). Importantly, this same protocol can be adapted to any RNA modification detectable by RNA-seq analysis for which the responsible protein is known.
AID/APOBEC 酶家族是作用于 DNA 和 RNA 的胞嘧啶脱氨酶。在 APOBEC 中，作用于 RNA 的最具特征性的家族成员是酶 APOBEC1。APOBEC1 介导的 RNA 编辑在脂质代谢和维持大脑内稳态中起着关键作用。编辑可以很容易地在 RNA-seq 数据中被检测到，因为相对于参考，胞嘧啶到胸腺嘧啶（C-to-T）的变化。然而，相对于参考，还有许多其他的碱基转换来源，如 PCR 错误、SNP，甚至是突变型 APOBECs 的 DNA 编辑。此外，APOBEC1 在不同的细胞类型中表现出不同的活性，相对于哪些转录本被编辑以及编辑的程度。在考虑这些潜在的误差和可变性来源时，野生型 APOBEC1 样本和具有 APOBEC1 敲除的匹配对照之间的 RNA-seq 比较是区分由 APOBEC1 编辑的真实位点的可靠方法。在这里，我们详细介绍了一种研究 APOBEC1 RNA 编辑的方法，特别是在鼠巨噬细胞系 RAW 264.7 中。我们的方法涵盖了使用 CRISPR/Cas9 系统产生 APOBEC1 敲除细胞系，直至实验验证和编辑位点的定量（我们讨论了一种最近发表的算法（称为 MultiEditR），该算法允许从 Sanger 测序中检测和定量 RNA 编辑）。重要的是，该方案可以适应任何可通过 RNA-seq 分析检测到的 RNA 修饰，而负责该修饰的蛋白质是已知的。
