Potential application of synthetic biology in disease information recording and real-time monitoring

doi:10.12211/2096-8280.2022-058

Abstract

Abstract:

The ability to change information stored with genome in real time, efficiently, and dynamically is a major technological advance for in situ studies of cell biology and biology as well to control cell phenotype, and monitor disease progression. Since it was first used for mammalian gene editing, CRISPR-Cas technology has been widely used in research, medicine development and industrial production. In addition to indel mutations induced by Cas9 activity, recent advances in CRISPR-Cas have enabled DNA or RNA base to be edited more efficiently, and synthetic biologists are developing devices for information storage by harnessing the versatilities of CRISPR-Cas-based tools and gene circuits to engineer cells with modifications. DNA has a strong ability to store information that is stable for thousands of years. Key technology for monitoring cell signal change and behavior coordination is to use DNA as the recorder of molecular events in the body, which can transfer transient signals in cells into sustainable reactions, and store them permanently. With this key technology, researchers could get an in-depth understanding on transformation from genotype to phenotype in health and disease states, drug reactions with diseases in clinical trials for patients, and environmental changes associated with activities of human being. The goal of synthetic biology is to engineer cells with genetic circuits for new biological functions. CRISPR-Cas based tools are useful in developing genetic circuits because they can be easily repurposed by designing complementary gRNAs that interfere or act on any arbitrary nucleic acid sequence of interest. Although they are still in their infancy, CRISPR-Cas based tools are gaining popularity in encoding biological memory and tracing and forwarding genetic screening for each formed lineage. In this article, we summarize the progress and application of synthetic biology in DNA storage and real-time monitoring in cell, as well as the advantages of the CRISPR-Cas system processes and records of information in living cells. Finally, we highlight their prospects and challenges in research on diseases and treatments.

Key words: synthetic biology, DNA store, real-time monitoring and recording systems in cell, disease information records, CRISPR-Cas systems

摘要：

关键词: 合成生物学, DNA存储, 细胞实时监测记录系统, 疾病信息记录, CRISPR-Cas系统

CLC Number:

MA Mengdan, LIU Yuchen. Potential application of synthetic biology in disease information recording and real-time monitoring[J]. Synthetic Biology Journal, 2023, 4(2): 301-317.

马孟丹, 刘宇辰. 合成生物学在疾病信息记录与实时监测中的应用潜力[J]. 合成生物学, 2023, 4(2): 301-317.

Figures/Tables 8

Table 1 Summary for real-time monitoring and recording systems in cell

记录装置系统		种群分布vs单细胞记录	写入周期	记录能力	发生顺序	持续时间	灵敏度	保真度
双稳态开关		种群	短	一般	否	是	低	低
DNA重组酶技术		种群	长	一般	否	否	低	低
ssDNA编辑技术	HiSCRIBE^[25]	种群	长	强	否	是	一般	高
CRISPR系统	Record-seq^[38]	种群	长	强	是	是	高	高
	CAMERA^[39]	单细胞	长	强	是	是	高	较高
	DNA typewriter^[40]	种群	长	强	是	是	高	高
	LINNAEUS^[41]	种群	短	弱	否	否	一般	低
	mSCRIBE^[42]	单细胞	长	强	是	是	高	高
	iTracer^[43]	单细胞	长	强	是	是	高	高
	DOMINO^[44]	种群	长	强	否	否	一般	一般

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

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

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

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

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

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

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.

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