Department of Genome Sciences, University of Washington, Seattle, WA, USA.
Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA.
Nat Protoc. 2024 Oct;19(10):2833-2862. doi: 10.1038/s41596-024-01003-0. Epub 2024 Jun 6.
Recording molecular information to genomic DNA is a powerful means of investigating topics ranging from multicellular development to cancer evolution. With molecular recording based on genome editing, events such as cell divisions and signaling pathway activity drive specific alterations in a cell's DNA, marking the genome with information about a cell's history that can be read out after the fact. Although genome editing has been used for molecular recording, capturing the temporal relationships among recorded events in mammalian cells remains challenging. The DNA Typewriter system overcomes this limitation by leveraging prime editing to facilitate sequential insertions to an engineered genomic region. DNA Typewriter includes three distinct components: DNA Tape as the 'substrate' to which edits accrue in an ordered manner, the prime editor enzyme, and prime editing guide RNAs, which program insertional edits to DNA Tape. In this protocol, we describe general design considerations for DNA Typewriter, step-by-step instructions on how to perform recording experiments by using DNA Typewriter in HEK293T cells, and example scripts for analyzing DNA Typewriter data ( https://doi.org/10.6084/m9.figshare.22728758 ). This protocol covers two main applications of DNA Typewriter: recording sequential transfection events with programmed barcode insertions by using prime editing and recording lineage information during the expansion of a single cell to many. Compared with other methods that are compatible with mammalian cells, DNA Typewriter enables the recording of temporal information with higher recording capacities and can be completed within 4-6 weeks with basic expertise in molecular cloning, mammalian cell culturing and DNA sequencing data analysis.
将分子信息记录到基因组 DNA 是一种强大的研究手段，可用于研究从多细胞发育到癌症进化等各种课题。基于基因组编辑的分子记录，细胞分裂和信号通路活性等事件会导致细胞 DNA 发生特定改变，从而在基因组上标记出关于细胞历史的信息，这些信息可以在事后读取。尽管基因组编辑已被用于分子记录，但在哺乳动物细胞中捕捉记录事件之间的时间关系仍然具有挑战性。DNA 打字机系统通过利用 Prime 编辑来促进对工程化基因组区域的顺序插入来克服这一限制。DNA 打字机包括三个不同的组件：DNA 带作为按顺序累积编辑的“底物”，Prime 编辑酶和 Prime 编辑引导 RNA，它们将插入编辑编程到 DNA 带上。在本方案中，我们描述了 DNA 打字机的一般设计考虑因素，逐步说明如何在 HEK293T 细胞中使用 DNA 打字机进行记录实验的步骤，以及用于分析 DNA 打字机数据的示例脚本（https://doi.org/10.6084/m9.figshare.22728758）。本方案涵盖了 DNA 打字机的两个主要应用：通过 Prime 编辑用可编程条形码插入记录顺序转染事件，以及在单个细胞扩增为多个细胞的过程中记录谱系信息。与其他适用于哺乳动物细胞的方法相比，DNA 打字机可以用更高的记录容量记录时间信息，并且在具有分子克隆、哺乳动物细胞培养和 DNA 测序数据分析的基本专业知识的情况下，在 4-6 周内即可完成。
