Nature sub-journal: Lou Chunbo/Wu Qiong team reconstructed high-resolution, programmable RNA regulatory network in living mammalian cells – Biological Research Area – Bio Valley

Source: Biological Exploration 2024-10-19 12:57

The RNA-IN/RNA-OUT gene circuit has the characteristics of high sensitivity, programmability, and single-base resolution; this circuit senses dynamic changes in RNA in living cells and directly converts them into transcriptional control instructions for specific genes. establish a strong relationship between them.

Lou Chunbo’s research group at the Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, collaborated with Wu Qiong’s research group at the School of Life Sciences, Tsinghua University. Nature Communications The journal published an article titled:High-resolution and programmable RNA-IN and RNA-OUT genetic circuit in living mammalian cells research paper.

This work realizes the reconstruction of any endogenous RNA regulatory network by constructing RNA-IN/RNA-OUT gene circuits with sensing and response functions in living mammalian cells; it also has the ability to sense point mutations in RNA sequences in living cells. For the first time, the single-point mutation sensing ability was increased from 1.5 times to 94 times; and instem cellsDifferentiation status sensing, cell endogenous progesterone anabolic pathway activation andtumor cellsIt has demonstrated a wide range of application potential in various cell therapy and gene therapy scenarios such as point mutation identification and selective elimination, and provides guidance for the determination of cell fate.AccurateControls provide a whole new toolbox.

The rapid development of single-cell technology and the progress of various cell atlas projects have provided a wealth of molecular labels for analyzing cell types and states in multiple spatial and temporal dimensions. RNA is a core mediator that determines the diversity of cell types and cell states. Dysregulation or mutations in its expression can lead to the evolution of various pathological cell states, including the occurrence of tumors. However, most current technologies are limited to RNA detection or tracing, and cannot directly convert RNA change signals into regulatory signals for cell status. Although the latest RNA sensors eToehold switch and ADAR switch have filled the gap in the field of RNA concentration sensing, they are not yet able to flexibly sense point mutations and have limitations such as poor designability or off-target toxicity. How to accurately, efficiently and programmably sense dynamic changes in broad-spectrum RNA concentration and sequence in living cells and regulate and manipulate specific target cells remains a key challenge facing the fields of life sciences and medicine.

This study proposes a new strategy aimed at developing highly sensitive, programmable, single-nucleotide resolution RNA sensing and response gene circuits in living cells, named RNA-IN/RNA-OUT. This circuit mainly includes three modules: the upstream RNA recognition and perception module responsible for endogenous RNA input (implementing RNA-IN), the downstream effector module responsible for endogenous RNA expression output (implementing RNA-OUT), and the processing responsible for RNA information presentation. module (Figure 1).

Figure 1. Schematic diagram of RNA-IN/RNA-OUT working principle

The research team first built a programmable RNA sensor, named CASP sensor, through a “recognition-activation” strategy, which exerts the sensing function of RNA dynamic signals to form an RNA-IN module. The CASP sensor consists of two parts: a programmable RNA-binding protein (DiCas7-11) and an effector protein (transcriptional activator CI434 and activation domain VP64). The CASP sensor follows the guidance of crRNA to activate protease activity and release effector proteins anchored in the cell membrane to achieve the purpose of transcriptional regulation. On the basis of precision design, in order to achieve sensitive response to a broad spectrum of RNA, the research team systematically debugged and optimized each component of the CASP sensor, successfully detecting endogenous expression as low as 8 transcripts per million transcripts. (TPM) (Figure 2).

Figure 2. Design and characterization of programmable CSAP sensor (RNA-IN)

In addition to abnormal expression of RNA, gene mutations also cause cancer,Blood vesselneurological diseases and other important causes of somatic diseases. However, there is often only a small difference in a single nucleotide mutation between wild-type and mutant RNA sequences, which is often difficult to detect and puts forward more challenging requirements for the sensing resolution of the RNA-IN module. However, in the CASP sensor, the key component of RNA sensing “DiCas7-11” has a high tolerance for single base mutations and is not sufficient to detect single point mutations. The research team systematically explored the critical point of DiCas7-11’s tolerance of base mismatches. Through the collaborative strategy of introducing auxiliary mutation sites, a single-base mismatch difference was formed between crRNA and the target RNA sequence, which was originally undetectable for the first time. The single point mutation RNA induction increased from 1.5 times to 94 times. As a result, the detection of single base mutations has been successfully achieved, and the sensing of RNA expression levels has been expanded to the sensing of sequence changes, which greatly enriches the recognition scope of the RNA-IN module. In particular, key genes in tumorsKRAS、TP53、BRAF、PIK3CA、EGFRIn the detection of representative point mutations, the CASP sensor demonstrated sensitive recognition ability (Figure 3).

Figure 3. Sensing and response of the cooperative CASP sensor to single nucleotide mutations (RNA-IN)

Furthermore, the research team connected the CASP sensor (RNA-IN) with the programmable dSpCas9-VPR endogenous activator (RNA-OUT) to form a complete RNA-IN/RNA-OUT gene circuit. This gene circuit is used to sense RNA at different expression levels in cells and to sense dynamically changing RNA inside cells when stimulated by environmental factors, ultimately achieving transcriptional activation of specific genes (Figure 4).

Figure 4. Design and optimization of RNA-IN/RNA-OUT gene circuits

Finally, the research team fully demonstrated the ultra-sensitive and flexible manipulation capabilities of endogenous RNA by the RNA-IN/RNA-OUT gene circuit in different cell types, including: 1) connecting continuously expressed RNA to activate progesterone. Endogenous biosynthetic metabolism network; 2) Dynamically monitor cell state changes in cell differentiation and transdifferentiation; 3) Selective killing of RNA by identifying characteristic point mutationspancreatic cancerandliver cancercells (Fig. 5).

Figure 5. Application expansion of RNA-IN/RNA-OUT gene circuits

In summary, the RNA-IN/RNA-OUT gene circuit has the characteristics of high sensitivity, programmability, and single-base resolution; this circuit senses dynamic changes in RNA in living cells and directly converts them into transcriptional regulatory instructions for specific genes. Establishing a strong association between any RNA has the potential to reconstruct the RNA regulatory network inside the cell and endow the cell with new biological functions. This circuit has broad application prospects in the fields of cell and gene therapy, cell reprogramming, and compound biosynthesis, providing innovative technical support for the manipulation of cell fate.

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