图1 双稳态开关设计［48］抑制物1抑制启动子1的转录，由诱导物1诱导；抑制物2抑制启动子2的转录，由诱导物2诱导

Fig. 1 Design for toggle switch[48]Repressors 1 and 2 inhibit transcription driven by Promoters 1 and 2, respectively, which is induced by Inducers 1 and 2 correspondingly

图2 三输入、16状态的RSM图示［55］（a）RSM机制。化学输入诱导重组酶的表达（来自输入质粒上的基因），由重叠和正交的重组酶识别位点组成的DNA登记表。不同的重组酶可以由不同的输入来控制。这些重组靶向多个正交对的同源识别位点（显示为三角形和半椭圆形），以催化反转（当这些位点是反对齐的）或切除（当这些位点是对齐的）。（b）登记表的设计是为每种身份和输入顺序采用不同的DNA状态。三种不同的输入（橙色、蓝色和紫色）由彩色箭头表示，每个箭头表示一个不同的重组酶。未重组的识别位点有阴影；重组的识别位点有轮廓

Fig. 2 Summary for three-input and 16-state RSM[55](a) RSM mechanism. A chemical input induces the expression of a recombinase encoded by a gene on the input plasmid, which modifies a DNA register with overlapping and orthogonal recombinase recognition sites. Specific recombinases can be controlled by corresponding inputs. Each of these recombinases can target multiple orthogonal pairs of their cognate recognition sites (shown as triangles and half-ovals) to catalyze inversion (when the sites are anti-aligned) or excision (when the sites are aligned). (b) The register is designed to adopt a specific DNA state for every identity and order of inputs. Three different inputs are represented by colored arrows (orange, blue, and purple), each of which expresses a specific recombinase. Unrecombined recognition sites are shown by solid symbols, and symbols without filling highlight recombined recognition sites

图3 SCRIBE实现了分布式基因组编码记忆［25］在输入存在的情况下，ssDNA（橙色曲线）从质粒携带的盒（灰色圆圈）中产生，并重组成基于序列同源性的特定基因组位点（橙色圆圈）。这就导致了精确突变的积累（绿色细胞中的星星），产生输入信号的大小和持续时间的相关函数

Fig. 3 SCRIBE-based distributed encoded memory at genome levels [25]In the presence of an input, ssDNAs (orange curved lines) are produced from a plasmid-borne cassette (gray circles) and recombined into specific genomic loci (orange circles) that are targeted on the basis of sequence homology. This results in the accumulation of precise mutations (stars in green cells) as a function of the magnitude and duration of exposure to the input

图4 从RNA中获取CRISPR间隔区的转录记录［59］（a）Record-Seq使用从食糖梭菌获得RNA的RT-Cas1-Cas2复合体将转录信息编码到质粒携带的CRISPR序列中。转录记录是通过直接从细胞内RNA获得CRISPR间隔区，然后通过FsRT-Cas1-Cas2的RT结构域逆转录RNA原间隔区来产生的。（b）提取质粒DNA，然后选择性扩增扩展CRISPR阵列（SENECA）和深度测序，进行转录历史的重建

Fig. 4 Transcriptional record of RNA extracted from CRISPR spacer acquisition[59](a) Record-seq uses the RNA-acquiring RT-Cas1-Cas2 complex from Fusicatenibacter saccharivorans to encode transcriptional information into plasmid-borne CRISPR arrays. The transcriptional record is generated by CRISPR spacer acquisition directly from intracellular RNAs followed by reverse transcription of RNA protospacers through the RT domain of FsRT-Cas1-Cas2. (b) Extraction of plasmid DNA followed by the selective amplification of expanded CRISPR arrays (SENECA) and deep sequencing enable the reconstruction of transcriptional histories

图5 CAMERA在细菌和哺乳动物细胞中的多路模拟细胞记录［39］CAMERA 1将刺激记录为相互排斥的DNA序列比例的变化。CAMERA 2使用碱基编辑器记录单核苷酸变化时信号的持续时间或振幅。这两个系统都可以复用，独立记录多个事件，包括暴露于抗生素、营养物质、病毒、光以及Wnt信号

Fig. 5 Multiple analog of cellular recording by CAMERA systems in bacteria and mammalian cells[39]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 repeatedly used to independently record multiple events, including exposure to antibiotics, nutrients, viruses, and light, as well as Wnt signaling

图6 用DNA typewriter进行序列基因组编辑［40］（a）Type guide上连续两个编辑序列件的示意图，它随着每个编辑序列的位置移动。DNA带由CRISPR-Cas9靶点（灰色方框）的串联序列组成，除第一个外，所有的靶点在其5′端被截断，因此不起作用。5 bp的插入包括一个2 bp的pegRNA特异条形码以及一个3 bp的key来激活下一个单体。因为基因组编辑在这个系统中是按顺序的，所以记录事件的时间顺序可以简单地通过它们沿着序列的物理顺序来读出。（b）用DNA typewriter引导编辑的示意图，引导编辑识别CRISPR-Cas9目标，并使用pegRNA指定的编辑对其进行修改。对于DNA typewriter，插入编辑序列会在随后的单体上生成新的编辑目标。（c）DNA typewriter顺序记录原理图，单个pegRNA与PE2酶一起，可以是事件驱动或者结构性表达的

Fig. 6 Sequential genome editing with DNA typewriter[40](a) Schematic of two successive editing events at the type guide, which shifts in position with each editing event. The DNA tape consists of a tandem array of CRISPR–Cas9 target sites (grey boxes), all but the first of which are truncated at their 5′ ends and therefore inactive. The 5-bp insertion includes a 2-bp pegRNA-specific barcode as well as a 3-bp key that activates the next monomer. Because genome editing is sequential in this scheme, the temporal order of recorded events can simply be read out by their physical order along the array. (b) Schematic of prime editing with DNA typewriter. Prime editing recognizes a CRISPR–Cas9 target and modifies it with the edit specified by the pegRNA. With DNA typewriter, an insertional editing event generates a new prime editing target at the subsequent monomer. (c) Schematic of ordered recording via DNA typewriter. Individual pegRNAs are potentially event-driven or constitutively expressed, together with the PE2 enzyme

图7 基于CRISPR-Cas9的信号导体的设计［70］重新设计的sgRNAs用来灭活（a）或激活（b）基因表达的链置换机制的说明。sgRNA的反义序列显示为蓝色，适配子茎显示为黄色。在没有信号A/B的情况下，sgRNA的引导区在反义茎内配对，sgRNA处于关闭状态。在信号A/B的存在下，重新设计的sgRNA的构象被切换到“ON”状态。在这种状态下，sgRNA的导向区与其目标DNA结合，从而分别通过dCas9-VP64融合蛋白和dCas9蛋白启动和关闭信号B/A的产生

Fig. 7 Design of the CRISPR-Cas9-based signal conductor to link one signal with another[70]General illustration of the strand-displacement mechanism by which the redesigned sgRNA acts to deactivate (a) or activate (b) gene expression. The antisense sequence of the sgRNA is shown in blue, and the aptamer stem is shown in yellow. In the absence of signal A/B, the guide region of sgRNA is paired within the antisense stem and the sgRNA is in the 'off' state. In the presence of signal A/B, the conformation of the redesigned sgRNA is switched to the 'on' state. In this state, the guide region of the sgRNA binds to its target DNA, and thus turns the production of signal B/A on and off through the dCas9-VP64 fusion protein and the dCas9 protein, respectively.