Writing a cell's history in its DNA Recording cellular events could advance our understanding of cellular history and responses to stimuli. The construction of intracellular memory devices, however, is challenging. Tang and Liu used Cas9 nucleases and base editors to record amplitude, duration, and order of stimuli as stable changes in both genomic and extrachromosomal DNA content (see the Perspective by Ho and Bennett). The recording of multiple stimuli—including exposure to antibiotics, nutrients, viruses, and light, as well as Wnt signaling—was achieved in living bacterial and human cells. Recorded memories could be erased and re-recorded over multiple cycles. Science, this issue p. eaap8992; see also p. 150 Base editors and CRISPR nucleases generate “cell data recorders” that enable durable, analog, rewritable recording of multiple stimuli. INTRODUCTION The stable recording of cellular events has the potential to advance our understanding of a cell’s history and how cells respond to stimuli. However, the construction of intracellular memory devices that record a history of cellular events has proven challenging. RATIONALE We developed two CRISPR-mediated analog multi-event recording apparatus (CAMERA) systems that record cellular events as durable changes in the DNA of bacteria or mammalian cells. In CAMERA 1, Cas9 nucleases are used to shift the ratio of two recording plasmids, and signals are recorded in the form of plasmid ratios. Writing in CAMERA 2 uses base editors to produce single-base modifications at designated positions of plasmid or genomic DNA. Both Cas9 nucleases and base editors can be programmed to target multiple DNA sequences with different guide RNAs, and both are known to function across many cell types. These features enable CAMERA to serve as a multiplexable, analog, rewritable intracellular recording system. RESULTS We demonstrate that the ratio of the recording plasmid pair in CAMERA 1 can be stably maintained in bacteria over 144 hours and a dilution ratio of 1017. By using a writing complex of the Cas9 nuclease and a guide RNA to selectively target one of the recording plasmids, we can cause this plasmid ratio to shift in a dose-dependent manner. The presence or absence of a stimulus is recorded in CAMERA 1 by linking to the expression of the writing complex. The analog format of CAMERA 1 enables recording of signal amplitude over a known time scale, or recording of the duration of a signal of known strength. Two resetting methods enable cells harboring CAMERA 1 to function over repeated cycles of recording and erasing. CAMERA 2 uses base editors to record stimuli of interest as permanent single-base modifications in cellular DNA. Predictable and dose-dependent accumulation of base editing was observed over 68 generations in bacteria. CAMERA 2 achieved analog recording of multiple stimuli of interest, including exposure to antibiotics, nutrients, viruses, and light. When recording to a high-copy plasmid, CAMERA 2 provides reliable readout by sequencing only 10 to 100 cells and can record event order using an overlapping guide RNA design. CAMERA 2 also functions in human cells by recording stimuli to safe-harbor genomic loci. We show that CAMERA 2 can be multiplexed, such that two responsive guide RNA expression cassettes can be used to record the presence of two exogenous small molecules in mammalian cells. Finally, we demonstrated CAMERA 2 recording of Wnt signaling, a crucial endogenous mammalian signaling pathway, as a permanent change in genomic DNA in human cells by placing the expression of the writing complex under the control of a Wnt-responsive promoter. CONCLUSION Base editors and CRISPR nucleases were used to create “cell data recorders” that enable durable, analog recording of stimuli and cell states. CAMERA systems are sensitive, multiplexable, resettable, and compatible with both bacteria and mammalian cells, and thus may be useful for applications such as recording the presence of extracellular and intracellular signals, mapping cell lineage, and constructing cell state maps. Multiplexed analog cellular recording by CAMERA systems in bacteria and mammalian cells. CAMERA 1 records stimuli as changes in the ratio of mutually exclusive DNA sequences. CAMERA 2 uses base editors to record the duration or amplitude of signals as single-nucleotide changes. Both systems can be multiplexed to independently record multiple events, including exposure to antibiotics, nutrients, viruses, and light, as well as Wnt signaling. We present two CRISPR-mediated analog multi-event recording apparatus (CAMERA) systems that use base editors and Cas9 nucleases to record cellular events in bacteria and mammalian cells. The devices record signal amplitude or duration as changes in the ratio of mutually exclusive DNA sequences (CAMERA 1) or as single-base modifications (CAMERA 2). We achieved recording of multiple stimuli in bacteria or mammalian cells, including exposure to antibiotics, nutrients, viruses, light, and changes in Wnt signaling. When recording to multicopy plasmids, reliable readout requires as few as 10 to 100 cells. The order of stimuli can be recorded through an overlapping guide RNA design, and memories can be erased and re-recorded over multiple cycles. CAMERA systems serve as “cell data recorders” that write a history of endogenous or exogenous signaling events into permanent DNA sequence modifications in living cells.

中文翻译：

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细菌和哺乳动物细胞中的可重写多事件模拟记录


在DNA中写入细胞的历史记录细胞事件可以增进我们对细胞历史和对刺激的反应的理解。然而，细胞内存储设备的构建具有挑战性。 Tang 和 Liu 使用 Cas9 核酸酶和碱基编辑器记录刺激的幅度、持续时间和顺序，作为基因组和染色体外 DNA 含量的稳定变化（参见 Ho 和 Bennett 的观点）。在活细菌和人类细胞中实现了多种刺激的记录，包括接触抗生素、营养物质、病毒和光以及 Wnt 信号传导。记录的记忆可以在多个周期内被擦除和重新记录。科学，本期第 14 页。 eaap8992；另见 p. 150 个碱基编辑器和 CRISPR 核酸酶生成“细胞数据记录器”，能够对多种刺激进行持久、模拟、可重写的记录。简介 细胞事件的稳定记录有可能增进我们对细胞历史以及细胞如何响应刺激的理解。然而，事实证明，构建记录细胞事件历史的细胞内存储装置具有挑战性。基本原理我们开发了两种 CRISPR 介导的模拟多事件记录装置 (CAMERA) 系统，可将细胞事件记录为细菌或哺乳动物细胞 DNA 的持久变化。在CAMERA 1中，Cas9核酸酶用于改变两个记录质粒的比率，并以质粒比率的形式记录信号。 CAMERA 2 中的写入使用碱基编辑器在质粒或基因组 DNA 的指定位置产生单碱基修饰。 Cas9 核酸酶和碱基编辑器都可以编程为具有不同指导 RNA 的多个 DNA 序列，并且已知两者都可以在多种细胞类型中发挥作用。 这些功能使 CAMERA 能够充当多路复用、模拟、可重写的细胞内记录系统。结果我们证明，在稀释倍数为1017的情况下，CAMERA 1中记录质粒对的比例可以在细菌中稳定维持144小时以上。通过使用Cas9核酸酶和引导RNA的写入复合物，选择性地靶向其中一个记录质粒质粒，我们可以使该质粒比例以剂量依赖性方式变化。通过链接到书写复合体的表达，将刺激的存在或不存在记录在相机 1 中。 CAMERA 1 的模拟格式能够记录已知时间范围内的信号幅度，或记录已知强度信号的持续时间。两种重置方法使包含 CAMERA 1 的单元能够在记录和擦除的重复循环中发挥作用。 CAMERA 2 使用碱基编辑器将感兴趣的刺激记录为细胞 DNA 中的永久单碱基修饰。在细菌中观察到超过 68 代的可预测且剂量依赖性的碱基编辑积累。 CAMERA 2 实现了对多种感兴趣刺激的模拟记录，包括接触抗生素、营养物质、病毒和光线。当记录到高拷贝质粒时，CAMERA 2 通过仅对 10 至 100 个细胞进行测序来提供可靠的读数，并且可以使用重叠向导 RNA 设计记录事件顺序。 CAMERA 2 还在人类细胞中通过记录对安全港基因组位点的刺激来发挥作用。我们证明 CAMERA 2 可以被多重化，这样两个响应性引导 RNA 表达盒可用于记录哺乳动物细胞中两种外源小分子的存在。 最后，我们通过将写入复合物的表达置于 Wnt 响应启动子的控制下，证明了 CAMERA 2 记录的 Wnt 信号传导（一种重要的内源哺乳动物信号传导途径）作为人类细胞中基因组 DNA 的永久性变化。结论 碱基编辑器和 CRISPR 核酸酶被用来创建“细胞数据记录器”，能够持久、模拟地记录刺激和细胞状态。 CAMERA 系统灵敏、可复用、可重置，并且与细菌和哺乳动物细胞兼容，因此可用于记录细胞外和细胞内信号的存在、绘制细胞谱系和构建细胞状态图等应用。 CAMERA 系统在细菌和哺乳动物细胞中进行多重模拟细胞记录。 CAMERA 1 将刺激记录为互斥 DNA 序列比例的变化。 CAMERA 2 使用碱基编辑器将信号的持续时间或幅度记录为单核苷酸变化。两个系统都可以复用以独立记录多个事件，包括接触抗生素、营养物质、病毒和光以及 Wnt 信号传导。我们提出了两种 CRISPR 介导的模拟多事件记录装置 (CAMERA) 系统，该系统使用碱基编辑器和 Cas9 核酸酶来记录细菌和哺乳动物细胞中的细胞事件。这些设备将信号幅度或持续时间记录为互斥 DNA 序列 (CAMERA 1) 比率的变化或单碱基修饰 (CAMERA 2)。我们实现了细菌或哺乳动物细胞中多种刺激的记录，包括接触抗生素、营养物质、病毒、光和 Wnt 信号传导的变化。当记录到多拷贝质粒时，可靠的读数只需要 10 到 100 个细胞。 刺激的顺序可以通过重叠引导RNA设计来记录，并且记忆可以在多个周期中被擦除和重新记录。 CAMERA系统充当“细胞数据记录器”，将内源或外源信号事件的历史写入活细胞中的永久DNA序列修改中。