RNA trans-splicing system based on split intron and application thereof

Abstract

The application discloses a kind of based on broken intron RNA trans-splicing system and application, belong to biotechnology field.The system includes the 5'RNA construct and 3'RNA construct of coding precursor RNA molecule, 5'RNA expressed by 5'RNA includes 5' exon, 5' intron and 5'EGS, 3'RNA construct expressed 3'RNA includes 3'EGS, IGS, 3' intron and 3' exon;5'EGS and 3'EGS complementary make 5' intron and 3' intron assemble to form complete intron, trigger splicing reaction, form mature mRNA including complete exon.The RNA trans-splicing system provided in the application greatly improves the RNA trans-splicing efficiency by fusing EGS sequence on the broken intron, and the trans-splicing system can be applied to gene regulation research, RNA sensor and the construction of biological computing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an RNA trans-splicing system based on broken introns and its applications. Background Technology

[0002] A long-held vision in synthetic biology research has been to design complex synthetic biological circuits as easily as electronic circuits using modular gene elements. However, the limited number of orthogonal elements currently available for assembly, the unpredictable behavior of gene circuits, and the metabolic burden that large-scale gene circuits place on cells make it difficult to scale up the size and complexity of gene circuits.

[0003] RNA-level gene regulatory elements offer an opportunity to solve this problem: due to the high programmability of RNA molecules, predictable base pairing mechanisms, and relatively clear thermodynamic laws, it is possible to design RNA element sequences using computers and predict the behavior of gene circuits. Moreover, RNA-based regulatory responses are faster and have a smaller genetic footprint, making them an excellent choice for constructing complex gene circuits. Currently, RNA regulatory elements such as pivot switches (Green, AA, Silver, PA, Collins, JJ & Yin, P. Toehold Switches: de-novo-designed regulators of gene expression. Cell 159, 925–939 (2014)) have been applied in fields such as biocomputing, in vitro disease detection, and metabolic engineering.

[0004] However, current RNA regulatory element design still has some drawbacks: First, many RNA elements rely on endogenous regulatory mechanisms within the host cell (such as mRNA translation and RNA degradation) to function, or depend on the assistance of protein elements. This leads to interference with gene circuit behavior by endogenous pathways within the host cell, as well as resource competition between gene circuits. Second, many RNA elements (such as pivot switches) alter the protein sequence of the target gene, causing unpredictable effects on protein activity. Finally, current RNA elements primarily utilize stem-loop structures for cis-regulation. Optimizing stem-loop structures requires extensive trial and error to avoid RNA misfolding, which can easily lead to leakage problems.

[0005] Group I introns are a class of RNA elements with autocatalytic activity. Under natural conditions, group I introns catalyze cis-splicing post-transcriptional transcription, linking the upstream 5' exon to the downstream 3' exon to form complete messenger RNA (mRNA). Simultaneously, the intron itself is cleaved from the original RNA molecule. Broken group I introns can undergo trans-splicing, where the 5' intron of one RNA molecule can assemble with the 3' intron, catalyzing a splicing reaction.

[0006] Currently, broken introns have been applied in gene therapy, RNA detection, RNA origami, and other fields, but they have not yet been developed into RNA regulatory elements in gene circuits. Moreover, studies have reported that the splicing efficiency of class I introns in bacteria is extremely low (Olson, KE & Muller, UFAn in vivo selection method to optimize trans-splicing ribozymes. RNA 18, 581–589 (2012)), making it difficult to develop a trans-splicing system for RNA in bacteria. Summary of the Invention

[0007] The purpose of this invention is to design an efficient and editable RNA regulatory element based on the trans-splicing of first-class introns, and to apply it to gene regulation research, RNA sensors, and the construction of biological computing.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides an RNA trans-splicing system based on broken introns. The system includes a 5' RNA construct and a 3' RNA construct encoding a precursor RNA molecule. The 5' RNA expressed by the 5' RNA construct includes a 5' exon, a 5' intron, and a 5' EGS from the 5' end to the 3' end. The 3' RNA expressed by the 3' RNA construct includes a 3' EGS, an IGS, a 3' intron, and a 3' exon from the 5' end to the 3' end. The 5' exon and the 3' exon are two fragments of a complete exon after being broken at the 5' splice site. The 3' end of the 5' exon contains the 5' splice site. The 5' intron and 3' intron are two fragments of a type I intron after it breaks at the loop position. The 3' end of the 3' intron contains a 3' splice site. The 5' EGS contains a sequence complementary to the 3' EGS. The 5' EGS and 3' EGS are composed of random sequences of 5 to 50 nt. Their complementarity allows the broken 5' and 3' introns to assemble. The IGS contains a sequence a complementary to the 4th to 9th bases at the 5' end of the 3' exon, a base paired with the 5' splice site in the 5' exon, and a sequence b complementary to the 5 bases upstream of the 5' splice site. The complementary double-stranded structure triggers the splicing reaction.

[0010] The working principle of the above system is as follows: 5' RNA constructs and 3' RNA constructs are transformed into cells and expressed to obtain precursor RNA molecules 5' RNA and 3' RNA. The 5' EGS of 5' RNA and the 3' EGS of 3' RNA complement each other to form an EGS double strand, promoting the assembly of 5' and 3' introns. In the assembled complete intron, the artificially designed internal guide sequence (IGS) forms a double-stranded structure P1 with the 5' P1 region in the 5' exon (i.e., the five bases upstream of the splice site in the exon). At the same time, the IGS complements the 5' end of the 3' exon (i.e., downstream of the 3' splice site at the 3' end of the 3' intron) to form a double-stranded structure, triggering the splicing reaction. The splicing reaction connects the 3' exon sequence after the 3' splice site to the 5' splice site, forming a mature mRNA containing a complete exon.

[0011] In this invention, the 5' RNA construct and the 3' RNA construct are constructed in a plasmid vector and transformed into cells for expression. Both the 5' RNA and 3' RNA are driven by independent inducible expression systems. Specifically, the 5' RNA construct comprises an inducible expression system, a ribosome binding site coding sequence, and a 5' RNA coding sequence; the 3' RNA construct comprises an inducible expression system and a 3' RNA coding sequence. The 5' RNA has a ribosome binding site integrated upstream, and intracellular ribosomes bind to this site to translate the downstream mRNA.

[0012] This invention promotes the assembly of 5' and 3' introns by fusing complementary EGS sequences to the 3' end of the broken 5' intron and the 5' end of the 3' intron, thus significantly improving the efficiency of RNA trans-splicing. The 5' and 3' EGS sequences can be composed of complementary random sequences. When either 5' or 3' EGS exists alone, it maintains a single-stranded RNA state; when both are present simultaneously, they can complementarily pair to form a double-stranded RNA.

[0013] To optimize the design of EGS sequences, this invention tested the splicing efficiency of EGS with different lengths and sequences in bacteria. Preferably, the sequence length of the complementary part of 5'EGS and 3'EGS is 10-20 nt, under which the introns assembled have high trans-splicing activity.

[0014] Preferably, the 5'EGS consists of a spacer sequence and a sequence complementary to the 3'EGS from the 5' end to the 3' end, wherein the spacer sequence is composed of 6 nt of random bases. The spacer sequence separates the complementary pairing region in the EGS from the 5' intron, thereby preventing the complementary pairing of the EGS from affecting the correct folding of the intron.

[0015] Preferably, the sequence of the 3'EGS is as shown in any of SEQ ID NO.1 to 80.

[0016] Preferably, corresponding to the above 3'EGS sequence, the 5'EGS sequence is shown in SEQ ID NO.81~160.

[0017] In this invention, after 5' RNA and 3' RNA are assembled, a double-stranded structure is formed through sequence complementarity of IGS, triggering the splicing reaction. IGS, by being complementary to both the 5' and 3' ends of the 3' exon, facilitates the correct identification of splice sites. Furthermore, it brings the 5' and 3' splice sites closer together in the RNA spatial structure, improving splicing efficiency. Preferably, the IGS, from the 5' end to the 3' end, consists of sequence a, complementary to the 5' end of the 3' exon, consecutive UUU bases, bases paired with the 5' splice site, and sequence b, complementary to the 5' P1 region in the 5' exon.

[0018] Preferably, the first type of intron can be, but is not limited to, some or all of the TT, BnrdE, BnrdF, Thy, Td, NrdB, SunY and NrdE2 introns. This invention provides eight introns with high trans-splicing activity. The TT intron is derived from *Tetrahymena thermophila*, and its nucleotide sequence is shown in SEQ ID NO. 161. The BnrdE and BnrdF introns are derived from the bnrdE and bnrdF genes of *Bacillus subtilis* SPβ phage, and their nucleotide sequences are shown in SEQ ID NO. 162 and SEQ ID NO. 163, respectively. The Thy intron is derived from the thy gene of *Bacillus subtilis* phage β22, and its nucleotide sequence is shown in SEQ ID NO. 164. The Td, NrdB, and SunY introns are derived from the td, nrdB, and SunY genes of *Escherichia coli* T4 phage, and their nucleotide sequences are shown in SEQ ID NO. 165, SEQ ID NO. 166, and SEQ ID NO. 167, respectively. The NrdE2 intron is derived from the nrdE gene of *Staphylococcus aureus* Twort phage, and its nucleotide sequence is shown in SEQ ID NO. 168.

[0019] Preferably, the first type of intron breaks at ring 1, ring 6 or ring 9.

[0020] In this invention, the 5' splice site is selected from the coding region of an exon or the 5' untranslated region. To achieve modular regulation of gene expression, a broken intron is inserted into the splice site of the 5' untranslated region. The 5' RNA does not contain the coding sequence of an exon, while the downstream of the 3' RNA contains the complete exon sequence. Since the 3' RNA does not contain the ribosome binding site necessary for RNA translation, the gene cannot be expressed normally when the 3' RNA is expressed alone.

[0021] Preferably, the base at the 5' splice site is uracil, guanine, adenine, or cytosine. Accordingly, in the IGS, the base matching uracil is guanine or adenine, the base matching guanine is cytosine, the base matching adenine is uracil or guanine, and the base matching cytosine is adenine or guanine.

[0022] The trans-splicing system provided by this invention extends the 5' splicing site from U to all four bases A, G, C, and U, and its feasibility has been verified in living cells, greatly improving the flexibility of designing splicing systems.

[0023] Preferably, the 5' splice site is chosen at the second position of the start codon.

[0024] The base at the 3' splice site is guanine.

[0025] Preferably, the 5' RNA construct comprises an inducible expression system, an insulator, a spacer sequence 1, a ribosome binding site, a spacer sequence 2, a 5' RNA coding sequence, and a terminator.

[0026] Preferably, the 3'RNA construct comprises an inducible expression system, an insulator, a 3'RNA coding sequence, and a terminator.

[0027] In this invention, a suitable induction expression system, insulator, and terminator can be selected based on the host cell. When the host cell is *Escherichia coli*, the following systems can be selected, but are not limited to: Ara induction system, Rha induction system, AHL induction system, and aTc induction system.

[0028] Interval sequence 1 and interval sequence 2 can be composed of random sequences of 5 to 6 nt, used to separate the constituent elements.

[0029] This invention also provides the application of the above-mentioned RNA trans-splicing system based on broken introns in modular regulation research of gene expression. The application includes: firstly, constructing 5' RNA constructs and 3' RNA constructs, and then transferring them into host cells by chemical transformation to induce the expression of 5' RNA and 3' RNA; the complementarity of 5'EGS and 3'EGS promotes the assembly of 5' introns and 3' introns, catalyzing the splicing reaction to form mature mRNA containing complete exons;

[0030] When the exon encodes the target protein, the 5' splice site is selected in the 5' untranslated region or at the second position of the start codon;

[0031] When the exons encode transcription factors, the application also includes constructing a recombinant vector containing a promoter sequence that binds to the transcription factor and a target gene, and transferring it into a host cell; a transcription activator recruits RNA polymerase to initiate the transcription of the target gene, and a transcription repressor blocks the binding of RNA polymerase to inhibit the transcription of the target gene.

[0032] When the exon encodes the sgRNA in the CRISPR-dCas9 system, the 5' end of the 5' exon is inserted with spacer sequence A. The application also includes constructing a recombinant vector containing an inactivated Cas9 expression module and a transcription activator PspFΔHTH::λN22plus expression module, and transferring it into a host cell. The complete sgRNA generated by splicing will assemble with the inactivated Cas9 protein and PspFΔHTH::λN22plus, and specifically activate the target promoter under the guidance of spacer sequence A.

[0033] The above system allows for modular regulation of the expression of any target gene, where the gene expression regulation is not directly aimed at disease diagnosis and treatment. As one embodiment of the invention, the modular gene expression regulation system can be used for the dynamic regulation of metabolic pathways to improve fermentation yield. For example, it can use metabolite-induced promoters (such as salicylic acid-inducible promoters) to control the expression of 5' RNA and 3' RNA, thereby controlling the expression of exons encoding key enzymes in metabolic pathways.

[0034] As one embodiment of the present invention, the above-described sgRNA-based modular gene expression system can also be used to specifically regulate the expression of bacterial endogenous genes. By modifying the spacer sequence of the sgRNA, it can be targeted to specific regions in the genome to selectively activate or inhibit the expression of endogenous genes.

[0035] This invention also provides the application of the above-mentioned RNA trans-splicing system based on broken introns in the construction of RNA sensors. The application includes: constructing a 5' RNA construct and a 3' RNA construct, wherein the 3' EGS is complementary to the target mRNA in the cell to be tested, and the sequence length of the complementary part is greater than the sequence length of the complementary part between the 3' EGS and the 5' EGS; the exon is a reporter gene.

[0036] The reporter gene can be, but is not limited to, genes encoding fluorescent proteins, luciferases, pigment proteins, etc., that can generate detection signals.

[0037] The working principle of the RNA sensor described above is as follows: When no target mRNA is present in the test cell, 5'EGS and 3'EGS pair normally to form a double-stranded EGS, which promotes the assembly of 5' and 3' introns, catalyzes the splicing reaction, and forms mature mRNA containing complete exons. The reporter gene is then expressed to generate a detection signal. When the target mRNA is present in the test cell, 3'EGS pairs with the target RNA, causing 5'EGS and 3'EGS to fail to pair normally, RNA splicing cannot occur, and no detection signal can be generated.

[0038] Specifically, the above system requires complementary pairing between the target RNA and the 3'EGS to inhibit the splicing activity of the RNA trans-splicing system of the present invention. Therefore, the 3'EGS sequence is designed from a region with fewer secondary structures in the target mRNA sequence to ensure that the 3'EGS can effectively bind to the target mRNA. Preferably, the sequence length is 40-50 nt, and the length of the complementary part of the 5'EGS to the 3'EGS is 20 nt.

[0039] As one embodiment of the present invention, the above-mentioned RNA sensor can be used to monitor the expression of intracellular RNA in bacteria, for example, to detect the expression of bacterial antibiotic resistance genes.

[0040] This invention also provides the application of the above-mentioned RNA trans-splicing system based on broken introns in constructing biological computing circuits. The application includes: constructing constructs of two-input logic gates (AND gate, NAND gate, implied NOT gate, OR gate, implied gate, NOR gate) and multi-input logic gates (AND gate, NAND gate), transferring them into cells by chemical transformation, and inducing RNA expression using different combinations of inducers.

[0041] In one embodiment of the present invention, for dual-input AND gates and NAND gates, the exons encode transcription activators such as ECF20 and transcription repressors such as LmrA, respectively. The construct includes a 5' RNA construct, a 3' RNA construct, and a recombinant vector containing a promoter sequence that binds to the transcription factor and the target gene. The principle is that the target gene will only be activated (AND gate) or repressed (NAND gate) when both 5' RNA and 3' RNA are simultaneously induced to express.

[0042] In one embodiment of the present invention, for a two-input NOT gate whose exons encode a reporter gene, the construct includes a 5' RNA construct, a 3' RNA construct, and a construct encoding a trans-repressive RNA complementary to 5'EGS. The principle is as follows: the trans-repressive RNA can complementarily pair with 5'EGS; if 5'EGS and 3'EGS cannot properly complementarily pair, RNA splicing cannot occur, and therefore the target gene is not expressed.

[0043] In one embodiment of the present invention, for a dual-input OR gate, the exons encode a reporter gene, and the construct includes a 5' RNA construct, 3' RNA-1, and 3' RNA-2 constructs. The principle is that the 5' RNA contains two 5' EGS, which can respectively pair complementaryly with the 3' EGS in the two 3' RNAs, leading to splicing.

[0044] In one embodiment of the present invention, for a dual-input implication gate, the exons encode a reporter gene, and the construct includes a 5' RNA construct, 3' RNA-1, 3' RNA-2 constructs, and a construct encoding a trans-repressive RNA complementary to 3'EGS-2. The principle is that either 3' RNA-1 or 3' RNA-2 can undergo splicing by complementary pairing with 5' RNA, but inducing the expression of the trans-repressive RNA inhibits the complementary pairing of 3'EGS-2 and 5'EGS, preventing splicing and thus preventing the expression of the target gene.

[0045] In one embodiment of the present invention, for a dual-input NOR gate, the exons encode a reporter gene, and the construct includes a 5' RNA construct, a 3' RNA construct, a construct encoding a trans-repressive RNA complementary to 5'EGS, and a construct encoding a trans-repressive RNA complementary to 3'EGS. The principle is that inducing expression of either of the two trans-repressive RNAs can block the normal complementary pairing of 5'EGS and 3'EGS, resulting in RNA splicing failing and the target gene failing to be expressed.

[0046] In one embodiment of the present invention, for a three-input AND gate, the exons encode a reporter gene, and the constructs include an RNA-1 construct, an RNA-2 construct, and an RNA-3 construct. The principle is as follows: the 5'EGS-1 in RNA-1 pairs complementaryly with the 3'EGS-1 in RNA-2, leading to splicing; the 5'EGS-2 in RNA-2 pairs complementaryly with the 3'EGS-2 in RNA-3, also leading to splicing. These two splicing reactions produce intact exons, resulting in the expression of the target gene.

[0047] For a four-input AND gate, the constructs include the RNA-1 construct, the RNA-2 construct, the RNA-3 construct, and the RNA-4 construct.

[0048] In one embodiment of the present invention, when the exons encode transcription factors such as ECF20, the principle is as follows: 5'EGS-1 in RNA-1 pairs complementaryly with 3'EGS-1 in RNA-2, leading to splicing; 5'EGS-2 in RNA-2 pairs complementaryly with 3'EGS-2 in RNA-3, leading to splicing; 5'EGS-3 in RNA-3 pairs complementaryly with 3'EGS-3 in RNA-4, leading to splicing. These three splicing steps produce intact exons, activating the expression of the target gene.

[0049] In one embodiment of the present invention, for a four-input AND gate, when its exons encode the N-terminal ECF20-N-terminal M86 inteptide fusion protein and the C-terminal M86 inteptide-C-terminal ECF20 fusion protein, the principle is as follows: 5'EGS-1 in RNA-1 and 3'EGS-1 in RNA-2 are complementary, leading to splicing, and the N-terminal ECF20-N-terminal M86 inteptide fusion protein is expressed; 5'EGS-2 in RNA-3 and 3'EGS-2 in RNA-4 are complementary, leading to splicing, and the C-terminal M86 inteptide-C-terminal ECF20 fusion protein is expressed. The N-terminal ECF20-N-terminal M86 inteptide fusion protein and the C-terminal M86 inteptide-C-terminal ECF20 fusion protein undergo protein splicing catalyzed by the breakage of the inteptide M86, producing the complete ECF20 protein and activating the expression of the target gene.

[0050] In one embodiment of the present invention, for a four-input NAND gate, its exons encode an N-terminal LmrA-N-terminal SspGyrB inteptide fusion protein and a C-terminal SspGyrB inteptide-C-terminal LmrA fusion protein. The principle is as follows: 5'EGS-1 in RNA-1 pairs complementaryly with 3'EGS-1 in RNA-2, leading to splicing and expression of the N-terminal LmrA-N-terminal SspGyrB inteptide fusion protein; 5'EGS-2 in RNA-3 pairs complementaryly with 3'EGS-2 in RNA-4, leading to splicing and expression of the C-terminal SspGyrB inteptide-C-terminal LmrA fusion protein. The N-terminal LmrA-N-terminal SspGyrB inteptide fusion protein and the C-terminal SspGyrB inteptide-C-terminal LmrA fusion protein undergo protein splicing catalyzed by the breakage of the inteptide SspGyrB, producing the intact LmrA protein and inhibiting the expression of the target gene.

[0051] For the six-input AND gate, the constructs include a 5'RNA-1 construct, a 3'RNA-1 construct, a 5'RNA-2 construct, a 3'RNA-2 construct, a 5'RNA-3 construct, and a 3'RNA-3 construct. In one embodiment of the invention, its exons encode an N-terminal ECF20-M86 intima-peptide fusion protein, a C-terminal M86 intima-peptide-mid-ECF20-N-terminal sspDnaX intima-peptide fusion protein, and a C-terminal sspDnaX intima-peptide-C-terminal ECF20 fusion protein. The principle is as follows: 5'EGS-1 in 5'RNA-1 pairs complementary with 3'EGS-1 in 3'RNA-1, leading to splicing and expression of the N-terminal ECF20-M86 inteptide fusion protein; 5'EGS-2 in 5'RNA-2 pairs complementary with 3'EGS-2 in 3'RNA-2, leading to splicing and expression of the C-terminal M86 inteptide-mid-terminal ECF20-N-terminal sspDnaX inteptide fusion protein; 5'EGS-3 in 5'RNA-3 pairs complementary with 3'EGS-3 in 3'RNA-3, leading to splicing and expression of the C-terminal M86 inteptide-ECF20 fusion protein. These three fusion proteins undergo splicing catalyzed by the breakage of inteptides M86 and sspDnaX, producing the complete ECF20 protein and activating the expression of the target gene.

[0052] The aforementioned logic circuitry can be used to program bacteria to respond to multiple input signals, enabling intelligent bacterial therapy. For example, by replacing the induction system in the AND gate circuit with sensors related to the tumor environment (such as hypoxia sensors, pH sensors, lactate sensors, etc.) and replacing the target gene with the expression of therapeutic proteins (such as nanobodies), bacteria can specifically secrete therapeutic proteins at the tumor site, improving the safety of tumor bacterial therapy.

[0053] The beneficial effects of this invention are as follows:

[0054] (1) The RNA trans-splicing system provided by this invention can control the splicing of target RNA and modularly regulate the expression of target genes. This trans-splicing system is suitable for rigorously controlling gene expression and reducing gene leakage, because no single RNA strand can produce a complete translation product on its own. In addition, seamless RNA splicing does not modify the target RNA and protein sequences, providing an attractive option for regulating highly structured non-coding RNAs and proteins with important N-terminal residues (such as N-terminal degradation tags).

[0055] (2) The RNA trans-splicing system provided by this invention significantly improves RNA trans-splicing efficiency by fusing EGS sequences to broken introns and utilizing the complementarity of EGS sequences to promote the assembly of broken introns into complete introns. This method of using EGS to improve RNA splicing efficiency can be applied to other fields based on type I intron RNA splicing, such as improving the yield of circular RNA or improving the efficiency of RNA editing.

[0056] (3) Class I introns are widely distributed in many species. The RNA trans-splicing system provided by this invention has the potential to be applied to strains commonly used in clinical medicine or fermentation industry to promote smart medicine and biomanufacturing.

[0057] (4) The RNA trans-splicing system provided by this invention can detect mRNA expression in bacteria to identify antibiotic resistance in target bacteria. This RNA splicing system also holds promise for application in the field of nucleic acid detection, in the preparation of reagents for in vitro detection of viral RNA to diagnose diseases.

[0058] (5) The RNA trans-splicing system provided by this invention can perform complex biological computations. By breaking the target gene into multiple segments, it improves the computational power of a single target gene, enabling complex multi-input biological computation circuits to be constructed from a limited number of target genes. This biological computation circuit can be used to integrate signals from multiple sensors for designing intelligent bacterial therapies and bacterial sensors, improving their performance and safety. Attached Figure Description

[0059] Figure 1 This is a basic design diagram of RNA trans-splicing.

[0060] Figure 2 This is a schematic diagram of the trans-splicing construct of Example 1. Where a is a schematic diagram of the 5' RNA construct; b is a schematic diagram of the 3' RNA construct.

[0061] Figure 3Characterization of the EGS sequence of the trans-splicing system in *E. coli*. Here, a) is the fluorescence characterization of the trans-splicing system in *E. coli*; b) is the effect of different EGS lengths on the trans-splicing efficiency in *E. coli*; and c) is the effect of different EGS sequences on the trans-splicing efficiency in *E. coli*.

[0062] Figure 4 Characterization of the splice sites in the trans-splicing system. Where a is a schematic diagram of the trans-splicing system designed with different splice sites; b is a schematic diagram of the 5' splice site using different bases; c is the fluorescence characterization of the trans-splicing system at different splice sites; d is the fluorescence characterization of the 5' splice site using different bases.

[0063] Figure 5 The diagrams show the constructs of the transcription factor trans-splicing system. In the diagrams, a represents the trans-splicing constructs of transcription activator ECF20, transcription repressor LmrA, and PhlF; b represents the trans-splicing construct of transcription repressor BM3R1; c represents the trans-splicing construct of transcription activator hrpR; and d represents the trans-splicing construct of sgRNA in the CRISPR-dCas9 system.

[0064] Figure 6 The effects of the trans-splicing system on splicing sites are shown. a) shows the fluorescence characterization of splicing sites in the 5' untranslated region and the start codon; b) shows the fluorescence characterization of downstream genes encoding sfGFP replaced with mCherry and mTagBFP in the start codon trans-splicing system; c) shows the fluorescence characterization of different transcription factor genes regulated by the trans-splicing system; and d) shows the fluorescence characterization of sgRNA activity in different CRISPR-Cas systems regulated by the trans-splicing system.

[0065] Figure 7 This is a characterization of the target mRNA detection using the RNA splicing system in Example 2. Wherein a is a schematic diagram of the design for detecting target mRNA using the RNA splicing system; b is the fluorescence characterization of the mRNA for detecting fluorescent proteins and antibiotic resistance genes using the RNA splicing system; c is a schematic diagram of the construct for detecting target mRNA using the RNA splicing system.

[0066] Figure 8The following are orthogonal characterizations of the components of the reverse splicing system in Example 3. Wherein a is a schematic diagram of the design of the splicing system based on orthogonal EGS; b is the fluorescence characterization of the splicing system based on orthogonal EGS when simultaneously expressing 5' RNA and 3' RNA, or expressing 3' RNA alone; c is the orthogonal characterization of the splicing system based on orthogonal EGS; d is a schematic diagram of the design of the splicing system based on orthogonal P1; e is the fluorescence characterization of the splicing system based on orthogonal P1 when simultaneously expressing 5' RNA and 3' RNA, or expressing 3' RNA alone; f is the orthogonal characterization of the splicing system based on orthogonal P1; g is a schematic diagram of the design of the splicing system based on orthogonal breakpoints; h is the fluorescence characterization of the splicing system based on orthogonal breakpoints with or without fused EGS; i is the orthogonal characterization of the splicing system based on orthogonal breakpoints; j is a schematic diagram of the design of the splicing system based on orthogonal introns; k is the fluorescence characterization of the splicing system based on orthogonal introns with or without fused EGS; l is the orthogonal characterization of the splicing system based on orthogonal introns.

[0067] Figure 9 This document describes the design and fluorescence characterization of logic gates based on RNA splicing. Specifically: a) Design and fluorescence characterization of a two-input AND gate based on RNA splicing; b) Design and fluorescence characterization of a two-input NAND gate based on RNA splicing; c) Design and fluorescence characterization of a two-input implied NOT gate based on RNA splicing; d) Design and fluorescence characterization of a two-input OR gate based on RNA splicing; e) Design and fluorescence characterization of a two-input implied gate based on RNA splicing; f) Design and fluorescence characterization of a two-input NAND gate based on RNA splicing; g) Design and fluorescence characterization of a three-input AND gate based on RNA splicing; and h) Design and fluorescence characterization of a four-input AND gate based on RNA splicing.

[0068] Figure 10 This is a schematic diagram of a two-input logic gate construct. Where a is a schematic diagram of a construct containing a NOT gate; b is a schematic diagram of a construct containing an OR gate; c is a schematic diagram of a construct containing a gate; and d is a schematic diagram of a construct containing a NOR gate.

[0069] Figure 11 The diagrams show multi-input logic gate constructs. 'a' is a schematic of a three-input AND gate construct; 'b' is a schematic of a four-input AND gate construct.

[0070] Figure 12This document describes the design and fluorescence characterization of multi-input logic gates based on RNA and protein splicing. Specifically, a) is the design of a four-input AND gate based on RNA and protein splicing; b) is the fluorescence characterization of a four-input AND gate based on RNA and protein splicing; c) is the design of a four-input NAND gate based on RNA and protein splicing; d) is the fluorescence characterization of a four-input NAND gate based on RNA and protein splicing; e) is the design of a six-input AND gate based on RNA and protein splicing; and f) is the fluorescence characterization of a six-input AND gate based on RNA and protein splicing.

[0071] Figure 13 The diagrams show logic gate constructs based on RNA and protein splicing. In the diagrams, a is a four-input AND gate construct; b is a four-input NAND gate construct; and c is a six-input AND gate construct. Detailed Implementation

[0072] Based on the structure of class I introns, the de novo design of RNA secondary structures, and the design principles of gene circuits, this invention has conducted in-depth research on trans-splicing reactions based on class I introns, thereby completing a gene regulation system based on RNA splicing.

[0073] Specifically, this invention significantly improves RNA trans-splicing efficiency by fusing EGS sequences to both ends of broken introns. To optimize EGS sequence design, this invention tested the splicing efficiency of EGS sequences of different lengths and sequences in E. coli, determined the optimal EGS length to be 20 nt, and used the RNA secondary structure design software NUPACK to generate 56 EGS sequences, constructing RNA splicing libraries with different splicing efficiencies.

[0074] Furthermore, this invention verified the versatility of the trans-splicing design, finding that it works at different splice sites and with different base pairs. Subsequently, this invention applied the trans-splicing design to modular gene expression regulation, successfully controlling the activity of fluorescent proteins, transcription activators, transcription repressors, and sgRNA in the CRISPR-Cas system using this system.

[0075] Next, this invention designs an EGS sequence to complement the target mRNA sequence, thus realizing a trans-splicing system regulated by mRNA. This invention uses this system to detect fluorescent proteins and antibiotic resistance gene mRNAs expressed in *E. coli*, enabling the identification of bacterial antibiotic resistance.

[0076] Furthermore, this invention investigates the construction of biological computing circuits using an RNA trans-splicing system. Through the design of orthogonal EGS, orthogonal P1, orthogonal intron breakpoints, and orthogonal introns, this invention achieves the construction of an orthogonal RNA trans-splicing system. Subsequently, based on this trans-splicing system, this invention enables the construction of two-input AND gates, NAND gates, implied gates, implied NOT gates, OR gates, NOR gates, as well as three-input and four-input AND gates in *E. coli*. Next, this invention introduces a protein trans-splicing system, achieving the construction of four-input AND and NAND gates, as well as a six-input AND gate, based on RNA and protein trans-splicing.

[0077] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0078] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0079] Example 1: Gene expression regulation in Escherichia coli

[0080] Based on the structure of class I introns, the de novo design of RNA secondary structures, and the design principles of gene circuits, this invention has conducted in-depth research on trans-splicing reactions based on class I introns, thereby completing a gene regulation system based on RNA splicing.

[0081] like Figure 1 As shown, this illustrates the basic principle of the gene expression regulation system of this invention. The system comprises two precursor RNA molecules: 5' RNA and 3' RNA. The 5' RNA molecule includes a 5' exon, a 5' intron, and a 5' EGS; wherein the 3' end of the 5' exon contains a 5' splicing site—uracil nucleotide (U). The 3' RNA includes a 3' EGS, a 3' intron, and a 3' exon; wherein the 3' end of the 3' intron contains a 3' splicing site—guanine nucleotide (G).

[0082] The following is a detailed explanation using the example of trans-splicing regulation of fluorescent protein expression using the above gene regulation system in Escherichia coli.

[0083] I. Regulation of fluorescent protein (sfGFP) expression in Escherichia coli

[0084] 1-1. In this embodiment, a method for rapidly determining RNA trans-splicing activity using fluorescence was established by breaking the Tetrahymena thermophilaintron (TTintron) at the loop 1 (L1) position and inserting the broken intron at the s482 site of the sfgfp gene.

[0085] The steps for regulating fluorescent protein expression using trans-splicing in E. coli are as follows:

[0086] 1) Construct 5' RNA and 3' RNA constructs using Gibson Assembly, transform the 5' RNA and 3' RNA constructs into E. coli using chemical transformation, and induce the expression of 5' RNA and 3' RNA;

[0087] 2) The 5' EGS of 5' RNA and the 3' EGS of 3' RNA can complement each other to form an EGS double strand, which promotes the assembly of 5' introns and 3' introns;

[0088] 3) In the assembled complete intron, the artificially designed internal guide sequence (IGS) forms a double-stranded structure P1 with the 5' P1 region in the 5' exon (i.e., the five bases upstream of the splice site in the exon), triggering the splicing reaction. Figure 4 a) The splicing reaction can ligate the 3' exon sequence after the 3' splice site to the 5' splice site to form a mature mRNA containing complete exons;

[0089] 4) Mature mRNA is translated into fluorescent protein.

[0090] In this embodiment, the 5' RNA construct uses the pSB3K3 plasmid vector (https: / / parts.igem.org / Part:pSB3K3), and the sequence configuration of the 5' RNA construct is as follows: AHL-induced expression system - insulator - spacer sequence 1 - ribosome binding site (RBS) - spacer sequence 2 - 5' exon - 5' intron - 5' EGS - terminator (…). Figure 2 ).

[0091] In this embodiment, the 3' RNA construct uses the pSB1A3 plasmid vector (https: / / parts.igem.org / Part:pSB1A3), and the sequence configuration of the 3' RNA construct is: AHL-induced expression system-insulator-3'EGS-3' intron-3' exon-terminator (...). Figure 2 ).

[0092] The sequences of the above components are shown in Table 1, and the EGS sequence is shown in Table 3.

[0093] Table 1. Sequences of each component of the construct

[0094]

[0095]

[0096] In this experimental method, the expression of green fluorescent protein depends on the mature mRNA produced by RNA splicing; therefore, a higher green fluorescence value indicates a higher RNA splicing efficiency.

[0097] This embodiment used multiple control groups to test the performance of RNA splicing elements. For example... Figure 3 As shown in Figure a, bacteria that simultaneously express 5' RNA and 3' RNA fused with EGS (Right 1 and Right 2 are experimental groups fused with EGS1 and EGS2, respectively; the sequences of EGS1 and EGS2 are shown in Table 3) show higher green fluorescence than bacteria that express only 5' RNA (Left 2) or 3' RNA (Left 1), and have lower output levels in the group with inactivated introns (Left 3, the inactivated intron is formed by a mutation of nucleotide 237 of the normal 3' intron body (from G to A), and cannot undergo splicing reaction; its sequence is shown in Table 1) or without EGS (Left 4).

[0098] These results indicate that green fluorescence output depends on RNA trans-splicing, and the presence or absence of EGS determines the splicing activity of broken introns.

[0099] 1-2. To optimize the design of RNA trans-splicing, this embodiment investigated the effects of EGS length and EGS sequence on splicing activity. First, this invention studied the effect of EGS length (0, 5, 10, 15, 20, 25, 30, 40, 50 nt) on trans-splicing activity. Figure 3 b). EGS sequences of different lengths are provided in Table 2. The results show that 10nt EGS can already produce significant trans-splicing fluorescence, while 20nt EGS has the highest trans-splicing activity.

[0100] Table 2. EGS sequences of different lengths

[0101]

[0102] 1-3. This example investigated the effect of EGS sequences on trans-splicing activity. Fifty-six EGS sequences were synthesized (Table 3), and their fluorescence in E. coli was characterized in two states: spliceable state (i.e., simultaneous expression of 5' RNA and 3' RNA) and non-spliceable state (i.e., expression of 3' RNA alone).

[0103] The results are as follows Figure 3 As shown in c, all EGS showed extremely low fluorescence values ​​in the non-splicing state and significantly increased fluorescence in the splicing state. Among the 56 EGS sequences, 46 EGS had activation folds greater than 10, 32 EGS had activation folds greater than 100, and 8 sequences had activation folds greater than 1000. The two best-performing EGS sequences showed fluorescence activation of over 10,000 times.

[0104] Table 3. EGS Sequences

[0105]

[0106]

[0107]

[0108] The above results indicate that, as a gene regulatory element, the leakage expression of the broken intron is extremely low, the fluorescence activation factor is high, and it is highly programmable (i.e., the target gene expression level can be achieved by adjusting the length and sequence of the EGS sequence).

[0109] 1-4. Based on the above-mentioned efficient EGS design, this embodiment expands the original trans-splicing system in two aspects: 1) changing the position of the 5' splice site, that is, inserting the broken intron into different sites of the sfgfp gene; 2) changing the nucleotides of the 5' splice site, that is, changing the bases from uracil (U) to guanine (G), adenine (A), and cytosine (C).

[0110] 1) Introns fused with either EGS-1 or EGS-2 were inserted into nine sites in the sfgfp gene (sequence shown in Table 1, consisting of sfgfp gene s1-s482 and sfgfp gene s483-s756): 20, 99, 196, 368, 482, 519, 567, 602, and 648. Since RNA splicing typically uses T as the 5' splice site, T on the sfgfp gene sequence was chosen as the insertion site. Corresponding IGS sequences were designed based on the 5' P1 sequence at different sites. Figure 4 a) The composition of the 5' RNA and 3' RNA constructs is the same. Figure 2 As shown. Then, the 5' RNA and 3' RNA constructs were transformed into E. coli to characterize splicing efficiency, while as a control group, the 3' RNA construct and the empty plasmid were also transformed into E. coli.

[0111] The results are as follows Figure 4 As shown in c, the fluorescence of simultaneous expression of 5'RNA and 3'RNA was higher than that of the control group at all 9 sites, indicating that the trans-splicing design of the present invention is applicable to different splicing sites.

[0112] 2) The nucleotides at the 5' splice site were mutated, and the corresponding IGS region was mutated accordingly. Figure 4 b). The results showed that, in addition to the traditional G·U base pair, other base pairs such as C·A, A·U, G·C, U·A, C·G, and A·G could also catalyze the trans-splicing reaction to produce fluorescent signals. Figure 4 d). Therefore, the trans-splicing system of the present invention can extend the 5' splicing site from U to all four bases A, G, C, and U, which will greatly improve the flexibility of designing splicing systems and has great application potential in gene therapy and other fields.

[0113] II. Modular regulation of gene expression in Escherichia coli

[0114] This embodiment applies the RNA splicing system to the modular regulation of gene expression, that is, by replacing the exon sequences in the splicing system, the expression of any target gene can be easily regulated.

[0115] 2-1. In order to design a modular RNA splicing system, this embodiment inserts the broken intron into two splicing sites in the 5' untranslated region of the fluorescent protein ( ). Figure 6 (the -6, -1 sites in a) and a site in the start codon ( Figure 6 The 3' RNA contains two sites (a, b, c) and is fused with EGS-1 and EGS-2. When the broken intron is inserted into the splice site of the 5' untranslated region, the 5' RNA does not contain the exon coding sequence, while the downstream of the 3' RNA contains the complete exon sequence. Because the 3' RNA does not contain the ribosome binding site necessary for RNA translation, the gene cannot be expressed normally when the 3' RNA is expressed alone.

[0116] Fluorescence results as follows Figure 6 As shown in Figure a, in the ON state, both 5' and 3' RNA are expressed simultaneously; in the OFF state, 3' RNA is expressed alone. All three splicing sites showed at least 32-fold higher fluorescence in the ON state compared to the OFF state, and the activation fold at site 2 exceeded 10,000-fold. Site 2 also exhibited good modularity, with similar "ON-OFF" behavior observed after changing the downstream reporter genes from sfGFP to mCherry and mTagBFP2. Figure 6 b).

[0117] 2-2. Subsequently, the RNA splicing system was extended from fluorescent proteins to transcription factors. When using trans-splicing of transcription factors to regulate target expression in *E. coli*, the steps are as follows:

[0118] 1) Provide 5' RNA and 3' RNA constructs, transform the 5' RNA and 3' RNA constructs into E. coli, and induce the expression of 5' RNA and 3' RNA;

[0119] 2) The 5' EGS of 5' RNA and the 3' EGS of 3' RNA can complement each other to form an EGS double strand, which promotes the assembly of 5' introns and 3' introns;

[0120] 3) In the assembled complete introns, a catalytic splicing reaction is carried out to form mature mRNA containing complete exons;

[0121] 4) Mature mRNA is translated into transcription factors;

[0122] 5) Transcription factors bind to downstream promoters, transcription activators recruit RNA polymerase to initiate transcription of the target gene, and transcription repressors block the binding of RNA polymerase to inhibit transcription of the target gene.

[0123] The construct composition of trans-splicing of transcription activator ECF20 is as follows: Figure 5 As shown in figure a, the 5' RNA construct controlled by the arabinose (Ara) induction system and the 3' RNA construct controlled by the rhamnose (Rha) induction system are located on the plasmid vector pSB3T5 (https: / / parts.igem.org / Part:pSB3T5), and the reporter gene activated by ECF20 is located on the plasmid vector pSB4A3 (https: / / parts.igem.org / Part:pSB4A3).

[0124] Constructs of trans-splicing of transcription activator HrpR, such as Figure 5 As shown in c, the 5' RNA construct controlled by the AHL induction system, the 3' RNA construct controlled by the anhydrotetracycline (aTc) induction system, and the hrpS construct controlled by the Rha induction system (hrpS is a protein factor that needs to be expressed simultaneously with hrpR to cooperate in activating the target promoter) are located on the plasmid vector pSB1A3, and the reporter gene activated by hrpR is located on the plasmid vector pSB3K3.

[0125] The constructs of trans-splicing of transcriptional repressors LmrA and PhlF are the same Figure 5 As shown in a, the 5' RNA construct controlled by the arabinose (Ara) induction system and the 3' RNA construct controlled by the rhamnose (Rha) induction system are located on plasmid vector pSB3K3, while the reporter gene repressed by LmrA or PhlF is located on plasmid vector pSB4A3.

[0126] Constructs of trans-splicing of the transcriptional repressor BM3R1, such as Figure 5As shown in b, the 5' RNA construct controlled by the Ara induction system and the 3' RNA construct controlled by the Rha induction system are located on plasmid vector pSB3K3, while the reporter gene repressed by BM3R1 is located on plasmid vector pSB4A3.

[0127] Constructs of sgRNA trans-splicing in the CRISPR-dCas9 system, such as Figure 5 As shown in d. The 5' RNA construct controlled by the AHL induction system and the 3' RNA construct controlled by the Ara induction system are located on plasmid vector pSB3K3, while dCas9, PspFΔHTH::λN22plus and the reporter gene construct are located on plasmid vector pSB4A3.

[0128] The induction system and promoter sequences used in this embodiment are shown in Table 4. The exons, introns, and EGS of the transcription factor transsplicing construct are shown in Table 5.

[0129] Table 4. Induction system and transcription factor sequences in this embodiment.

[0130]

[0131]

[0132] manual

[0133]

[0134] Table 5. Exon, intron, and EGS sequences of the transcription factor trans-splicing construct in this embodiment.

[0135]

[0136]

[0137] Fluorescence results of transcription factor trans-splicing regulating gene expression are as follows: Figure 6 As shown in Figure c. In this experiment, the promoter, controlled by transcription factors, controls the expression of the green fluorescent protein gene (target gene). When 5' RNA and 3' RNA are expressed simultaneously, the transcription activators ECF20 and HrpR can upregulate the expression of the target gene by 315-fold and 65-fold, respectively, while the transcription repressors LmrA, PhlF, and BM3R1 can reduce the expression of the target gene by 81, 90, and 33-fold, respectively.

[0138] Similarly, the trans-splicing of this invention can regulate the activity of sgRNA in the CRISPR-dCas9 system. In the trans-splicing-controlled CRISPR system, the simultaneous expression of 5'sgRNA and 3'sgRNA activates sgRNA splicing. The spliced, intact sgRNA assembles with the inactivated Cas9 (dCas9) protein and transcription activator (PspFΔHTH::λN22plus), specifically activating the target promoter under the guidance of the spacer sequence. Figure 6 d). The inverse splicing controlled CRISPR system of the present invention has excellent modularity and can produce activation greater than 22 times for four different interval sequences (LEA1, LEB1, LEA3, LEB3, see Table 5).

[0139] Example 2: RNA sensing in Escherichia coli

[0140] The EGS in this invention can be programmed to detect intracellular mRNA. In this embodiment, trans-splicing is used in *E. coli* to detect the expression of target RNAs (reporter gene mCherry, antibiotic resistance genes ampR (ampicillin resistance) and cat (chloramphenicol resistance) mRNAs), and the steps are as follows:

[0141] 1) Provide constructs expressing 5' RNA, 3' RNA, and target RNA, with the construct composition as follows: Figure 7 As shown in c: the target gene controlled by the Rha induction system and the 3' RNA construct controlled by the AHL induction system are located on plasmid vector pSB1A3, and the 5' RNA construct controlled by the AHL induction system is located on plasmid vector pSB3K3.

[0142] The reporter gene mCherry sequence is available in accession number MN872303.1 (bases 6264 to 6974); the antibiotic resistance gene ampR (ampicillin resistance) sequence is available in accession number MK756311.1 (bases 2667 to 3527); and the antibiotic resistance gene cat (chloramphenicol resistance) sequence is available in accession number CP050522.1 (bases 4150783 to 4151442). The intron break is inserted at site s2 of the sfgfp gene; the sequence is shown in Table 1. The EGS sequence and the EGS-target RNA binding region sequence are shown in Table 6.

[0143] 2) The construct was transformed into E. coli, and the expression of 5' RNA and 3' RNA was induced;

[0144] 3) If there is no target RNA in E. coli, the 5'EGS and 3'EGS pair normally to form a double strand of EGS, which promotes the assembly of the 5' intron and the 3' intron;

[0145] 4) In the assembled complete intron, a catalytic splicing reaction occurs to form a mature mRNA containing complete exons. The mRNA is then translated into the fluorescent protein sfGFP.

[0146] 5) If the target RNA is present in E. coli, the 3'EGS pairing with the target RNA prevents the 5'EGS and 3'EGS from properly complementing each other, thus preventing RNA splicing. Figure 7 a).

[0147] Table 6. EGS sequence and target RNA sequence used by the trans-splicing RNA sensor in this embodiment.

[0148]

[0149]

[0150] like Figure 7 Figure b shows the fluorescence results of the trans-splicing RNA sensor of the present invention for detecting the expression of mRNA corresponding to the reporter gene mCherry, the antibiotic resistance genes ampR (ampicillin resistance), and cat (chloramphenicol resistance). The sensor designed in this invention can detect the expression of the mRNA corresponding to the above three genes, and the fluorescence signal decreases by more than 154 times when the target mRNA is present.

[0151] Therefore, this invention is of great significance for monitoring the expression of intracellular RNA in bacteria, such as for detecting antibiotic-resistant bacteria.

[0152] Example 3: Biological Computation in Escherichia coli

[0153] I. Constructing orthogonal reverse splice circuits

[0154] The programmability of EGS in this invention makes it possible to design orthogonal gene regulatory elements and biological computing circuits. Orthogonality refers to the ability of gene elements or gene circuits to function independently without interfering with each other. Good orthogonality (or low crosstalk) is a prerequisite for constructing high-order logic computing circuits and performing multiple gene regulation.

[0155] 1-1. To construct an orthogonal trans-splicing system, this embodiment first designed and validated an orthogonal EGS library containing 24 sequences (Table 7). The orthogonal EGS library was constructed using the multi-tube design program in NUPACK software. In this program, EGS binding was defined as a one-step reaction: in step 0, the two RNAs (5'EGS and 3'EGS) remain single-stranded; in step 1, the two RNAs bind to form a double strand. Therefore, to design the orthogonal system, each system has two step tubes (step 0 tube and step 1 tube) and one global crosstalk tube. Then, the orthogonal system was defined in Python using a loop, and EGS sequences with low global crosstalk were designed. Subsequently, the 5' RNA construct and 3' RNA construct of the orthogonal trans-splicing system were constructed, with sequence compositions identical to those of the previous model. Figure 2 As shown, the sequence configuration of the 5' RNA construct is: AHL-induced expression system - insulator - spacer sequence 1 - RBS - spacer sequence 2 - 5' exon - 5' intron - 5' EGS - terminator; the sequence configuration of the 3' RNA construct is: AHL-induced expression system - insulator - 3' EGS - 3' intron - 3' exon - terminator. In this embodiment, the sequences of the AHL-induced expression system, insulator, spacer sequence, RBS, intron, and terminator are as shown in Table 1. The broken intron is inserted at site s2 of the sfgfp gene, and the EGS sequence is provided in Table 7.

[0156] Table 7. Orthogonal EGS library sequences

[0157]

[0158]

[0159] like Figure 8 Figure b shows the characterization data for orthogonal EGS-O1 to EGS-O24 in *E. coli*. In the ON state, 5' RNA and 3' RNA are expressed simultaneously; in the OFF state, 3' RNA is expressed alone. For all EGS sequences, the fluorescence in the ON state is at least 380-fold higher than that in the OFF state. Crosstalk levels between EGS were characterized by co-transforming 5' RNA constructs fused with random 5' EGS and 3' RNA constructs fused with random 3' EGS into *E. coli* and inducing expression. Crosstalk levels can be derived from the ratio of the fluorescence level of a specific 5' EGS-3' EGS combination to the fluorescence level of that 3' EGS and its originally paired 5' EGS combination. This library exhibits good orthogonality with crosstalk levels below 16% (crosstalk levels exceeding 10% were observed only in 3' EGS-O22 and 5' EGS-O21). Figure 8 c).

[0160] 1-2. This embodiment extends the orthogonal splicing system from orthogonal EGS to orthogonal P1 ( Figure 8 d) Orthogonal break points ( Figure 8 g) and orthogonal introns ( Figure 8 j).

[0161] The P1 sequence, which showed low homology among the three sequences, exhibited a clear ON-OFF behavior. Figure 8 e) and good orthogonality ( Figure 8 f).

[0162] Introns were broken in the three ring regions (L1, L6, and L9). The splicing activity of the intron fused with EGS was 4738, 66, and 3689 times higher than that of the broken introns without EGS fusion. Figure 8 These splitting sites also showed good orthogonality, except for the noticeable crosstalk observed between 5'L9 and 3'L6 (h). Figure 8 i).

[0163] This embodiment identified and characterized eight introns with high trans-splicing activity (Table 8). Among these introns, BnrdE and BnrdF introns originated from the bnrdE and bnrdF genes of Bacillus subtilis SPβ phage, Thy intron originated from the td gene of Bacillus subtilis phage β22, Td, NrdB, and SunY introns originated from the td, nrdB, and SunY genes of Escherichia coli T4 phage, and NrdE2 intron originated from the nrdE gene of Staphylococcusaureus Twort phage.

[0164] Table 8. Orthogonal Intron Library Sequences

[0165]

[0166]

[0167]

[0168] like Figure 8 As shown in k, these are fluorescence data on the splicing efficiency of different fragmented introns in *E. coli*. Except for the SunY intron, the fluorescence of all fragmented introns fused with EGS-1 and EGS-2 was at least 66 times higher than that of fragmented introns fused with unfused EGS. These fragmented introns also exhibited good orthogonality, with all crosstalk levels below 6% when fused with the same EGS (EGS-1 or EGS-2, Table 3). Figure 8 This is the first orthogonally split intron library reported to date.

[0169] 1-3. This embodiment proposes that by combining orthogonal introns with orthogonal EGS, the library of the orthogonal inverse splicing system can be further expanded. Combining 8 orthogonal splicing introns and 2 orthogonal EGS (EGS-1 and EGS-2) doubles the size of the orthogonal library, expanding it from 8 to 16. Figure 8 (l). Although significant crosstalk was observed between EGS-1 and -2 in the TT and Thy introns, the maximum crosstalk of all other 254 3'RNA-5' RNA pair combinations was less than 7.4%.

[0170] II. Constructing a biological computing circuit based on RNA splicing

[0171] This embodiment demonstrates the use of an RNA trans-splicing system for constructing biological computing circuits.

[0172] 2-1. This embodiment constructs six two-input logic gates. These logic gates are based on three induction systems, Ara, Rha, and AHL, the sequences of which are provided in Table 4. The composition of these logic gate constructs is as follows: Figure 10 As shown, its component sequences are shown in Table 9.

[0173] Two-input AND gates and NAND gates use arabinose and rhamnose promoters to control the expression of 5' RNA and 3' RNA, respectively. Figure 9 a, b). The constructs and sequences of AND and NAND gates and the constructs of the transcription factor trans-splicing system (Example 1). Figure 5 The same applies. For the AND gate, it induces trans-splicing of ECF20 in both RNA strands, and activates fluorescence output 300-fold ( Figure 9 a) For NAND gates, the transcriptional repressor LmrA is produced to inhibit downstream GFP expression only when both RNAs are present. The dynamic range of the NAND gate constructed from the transcriptional repressors LmrA, PhlF, and BM3R1 is 24 to 181. Figure 6 c, Figure 9 b).

[0174] A NIMPLY gate was constructed by designing and synthesizing a trans-repressive RNA (trRNA) that is complementary to 5' RNA (as in Example 2). The NIMPLY gate construct is as follows: Figure 10 As shown in a. trRNA exhibits a strict inhibitory effect on splicing activity; inducing trRNA expression can suppress fluorescence output by up to 1986-fold ( Figure 9 c).

[0175] The OR gate is achieved by fusing two 5' RNAs, and its construct is as follows: Figure 10As shown in b. Both 3' RNAs can splice with the fused 5' RNA, thereby upregulating sfGFP expression by 286-fold. Figure 9 d).

[0176] An implying gate was constructed by utilizing one of the 3' RNAs in the trRNA repression or gate, achieving a dynamic range of 34-fold. Figure 9 e) Its construct composition is as follows Figure 10 c.

[0177] Two trRNA repression gates with 3'EGS and 5'EGS are designed to implement NOR gate logic. The construct is as follows: Figure 10 d. Any trRNA can suppress output fluorescence by at least 51-fold ( Figure 9 f).

[0178] 2-2. In order to scale up these biological circuits, the present invention increases the number of inputs to AND and NAND gates by inserting more introns into the target gene.

[0179] First, two pairs of fragmented introns fused with EGS-2 and EGS-1 were inserted into sites 2 and 482 of the sfgfp gene, and its mRNA was split into three fragments to construct a three-input AND gate (its construct composition is as follows). Figure 11 a). Compared to all "false" states, the three-input AND gate increases by 250 times in the "true" state. Figure 9 g).

[0180] Subsequently, three pairs of broken introns were inserted into sites 2, 144, and 491 of the ecf20 gene to construct four input AND gates (the constructs of which are as follows). Figure 11 (b) When four RNA molecules are expressed simultaneously, they splice to produce complete mRNA, which is then translated into the transcription factor ECF20. ECF20 can activate the expression of downstream GFP. Output GFP expression was observed to be 16-fold higher in the presence of the four inducible signals (Ara, Rha, AHL, and aTc) than in other cases. Figure 9 h).

[0181] Table 9. DNA sequences of logic gate construct components

[0182]

[0183]

[0184] III. Constructing biological computing circuits based on RNA and protein splicing

[0185] The RNA trans-splicing system of this invention can be combined with a protein splicing system to perform more complex single-layer multi-input logic computations. To demonstrate this logic computation capability, this embodiment introduces a protein splicing system based on fragmented inteins (Pinto, F., Thornton, EL & Wang, B. An expanded library of orthogonal splitinteins enables modular multi-peptide assemblies. Nat. Commun. 11, 1529 (2020)). The fragmented intein system comprises two peptide chains, an N-terminal peptide chain and a C-terminal peptide chain. The N-terminal peptide chain comprises an N-terminal target peptide chain and an N-terminal intein, and the C-terminal peptide chain comprises a C-terminal intein and a C-terminal target peptide chain.

[0186] When performing biocomputation using fragmented introns and fragmented intron peptides in E. coli, the steps are as follows:

[0187] 1) Provide 5' RNA and 3' RNA constructs (e.g.) Figure 11 ), and the 5' RNA and 3' RNA constructs were transformed into E. coli, and the expression of 5' RNA and 3' RNA was induced;

[0188] 2) The 5' EGS of 5' RNA and the 3' EGS of 3' RNA can complement each other to form an EGS double strand, which promotes the assembly of 5' introns and 3' introns;

[0189] 3) In the assembled complete intron, a catalytic splicing reaction occurs to form a mature mRNA containing complete exons. The mRNA is then translated into N-terminal and C-terminal peptide chains.

[0190] 4) The N-terminal intipeptide in the N-terminal peptide chain and the C-terminal intipeptide in the C-terminal peptide chain assemble into a complete intipeptide.

[0191] 5) Intended peptides catalyze protein splicing reactions, linking the N-terminal and C-terminal target peptide chains to form complete protein molecules (transcription factors);

[0192] 6) Transcription factors bind to downstream promoters, transcription activators recruit RNA polymerase to initiate transcription of the target gene, and transcription repressors block the binding of RNA polymerase to inhibit transcription of the target gene.

[0193] First, two pairs of fragmented introns are inserted into the N-terminal and C-terminal peptide chains, respectively, dividing the target ECF20 gene into four segments, each controlled by one of four inducible promoters (AHL, Rha, aTc, and Ara inducible promoters), to construct a four-input AND gate. The principle is as follows: Figure 12 a, its construct composition is as follows Figure 13a. When all four inducers are present, the four RNA strands undergo two RNA trans-splicing reactions to produce two mature mRNAs, which are translated into two peptide chains (N-terminal ECF20-N-terminal M86 inteptide and C-terminal M86 inteptide-C-terminal ECF20). These then undergo protein trans-splicing to produce the complete ECF20 transcription factor, which in turn activates the expression of downstream green fluorescent protein. The fluorescence intensity of this four-input AND gate in the presence of all four inducers is 172 times that in other states. Figure 12 b).

[0194] Subsequently, two pairs of broken introns were inserted into the N-terminal peptide chain (N-terminal LmrA-N-terminal SspGyrB intron) and the C-terminal peptide chain (C-terminal SspGyrB intron-C-terminal LmrA) of the transcriptional repressor factor LmrA, respectively, to construct a four-input NAND gate. The principle is as follows: Figure 12 c, its construct composition is as follows Figure 13 b. The fluorescence intensity of this four-input NAND gate is 80 times lower in the presence of all four inducers than in other states. Figure 10 d).

[0195] Finally, the transcription activator ECF20 was cleaved into three polypeptide chains using two pairs of orthogonal introns (M86 and sspDnaX): N-terminal ECF20-N-terminal M86 intron, C-terminal M86 intron-mid-terminal ECF20-N-terminal sspDnaX intron, and C-terminal sspDnaX intron-C-terminal ECF20. The three pairs of orthogonal cleavage introns were then inserted into the mRNAs encoding the three polypeptide chains to construct a six-input AND gate. The principle is as follows: Figure 12 e, whose building block composition is as follows Figure 13 c. The fluorescence level produced by this six-input AND gate in the presence of six RNAs (5'RNA-1, 5'RNA-2, 5'RNA-3, 3'RNA-1, 3'RNA-2, and 3'RNA-3) is 30 times higher than in other cases. Figure 12 f).

[0196] The fragmented intima peptide sequences used in this embodiment are shown in Table 10. The sequences of the construct components used in this embodiment are shown in Table 11.

[0197] Table 10. DNA sequences of logic gate construct components in this embodiment

[0198]

[0199]

[0200] Table 11. DNA sequences of logic gate construct components in this embodiment

[0201]

[0202]

[0203] The embodiments described above are merely specific examples of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention, such as selecting appropriate induction systems and output genes for different application scenarios. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. An RNA trans-splicing system based on broken introns, characterized in that, The system includes a 5' RNA construct and a 3' RNA construct encoding a precursor RNA molecule. The 5' RNA construct comprises an inducible expression system, a ribosome binding site coding sequence, and a 5' RNA coding sequence. The 3' RNA construct comprises an inducible expression system and a 3' RNA coding sequence. Both the 5' RNA and 3' RNA are expressed by independent inducible expression systems. The 5' RNA expressed by the 5' RNA construct includes a 5' exon, a 5' intron, and a 5' EGS from the 5' end to the 3' end; the 3' RNA expressed by the 3' RNA construct includes a 3' EGS, an IGS, a 3' intron, and a 3' exon from the 5' end to the 3' end. The 5' exon and the 3' exon are two fragments of a complete exon after being broken at the 5' splice site. The 3' end of the 5' exon contains a 5' splice site, and the bases of the 5' splice site are uracil, guanine, adenine, or cytosine. The 5' intron and the 3' intron are two fragments of a type I intron after being broken at loop 1, loop 6, or loop 9. The 3' end of the 3' intron contains a 3' splice site. The type I intron is any of the sequences shown in SEQ ID NO. 161~168. 5'EGS contains a sequence complementary to 3'EGS. Both 5'EGS and 3'EGS are composed of random sequences of 5 to 50 nt. Their complementarity enables the assembly of broken 5' and 3' introns. IGS contains sequence a, which is complementary to the 4th to 9th bases at the 5' end of the 3' exon, a base that pairs with the 5' splice site in the 5' exon, and sequence b, which is complementary to the 5 bases upstream of the 5' splice site. The complementary double-stranded structure triggers the splicing reaction.

2. The RNA trans-splicing system based on broken introns as described in claim 1, characterized in that, The sequence length of the complementary portion of 5'EGS and 3'EGS is 10~20nt; the 5'EGS consists of a spacer sequence and a sequence complementary to 3'EGS from the 5' end to the 3' end, and the spacer sequence consists of 6nt random bases.

3. The RNA trans-splicing system based on broken introns as described in claim 2, characterized in that, The 3'EGS sequence is shown in any of SEQ ID NO.1~80, and the corresponding 5'EGS sequence is shown in any of SEQ ID NO.81~160.

4. The RNA trans-splicing system based on broken introns as described in claim 1, characterized in that, The 5' splice site is selected from either the coding region or the 5' untranslated region of the exon.

5. The RNA trans-splicing system based on broken introns as described in claim 1 or 4, characterized in that, In IGS, the bases that match uracil are guanine or adenine, the bases that match guanine are cytosine, the bases that match adenine are uracil or guanine, and the bases that match cytosine are adenine or guanine.

6. The RNA trans-splicing system based on broken introns as described in claim 1, characterized in that, The 5' RNA construct consists of an inducible expression system, an insulator, spacer sequence 1, a ribosome binding site, spacer sequence 2, a 5' RNA coding sequence, and a terminator; the 3' RNA construct consists of an inducible expression system, an insulator, a 3' RNA coding sequence, and a terminator.

7. The application of the RNA trans-splicing system based on broken introns as described in any one of claims 1-6 in modular regulation research of gene expression, characterized in that, The application includes: firstly, constructing 5' RNA and 3' RNA constructs, which are then transferred into host cells via chemical transformation to induce the expression of 5' RNA and 3' RNA; the complementarity of 5'EGS and 3'EGS promotes the assembly of 5' introns and 3' introns, catalyzing splicing reactions to form mature mRNA containing complete exons; When the exon encodes the target protein, the 5' splice site is selected in the 5' untranslated region or at the second position of the start codon; When the exons encode transcription factors, the application also includes constructing a recombinant vector containing a promoter sequence that binds to the transcription factor and a target gene, and transferring it into a host cell; a transcription activator recruits RNA polymerase to initiate the transcription of the target gene, and a transcription repressor blocks the binding of RNA polymerase to inhibit the transcription of the target gene. When the exon encodes the sgRNA in the CRISPR-dCas9 system, the 5' end of the 5' exon is inserted with spacer sequence A. The application also includes constructing a recombinant vector containing an inactivated Cas9 expression module and a transcription activator PspFΔHTH::λN22plus expression module, and transfecting it into a host cell. The complete sgRNA generated by splicing will assemble with the inactivated Cas9 protein and PspFΔHTH::λN22plus, and specifically activate the target promoter under the guidance of spacer sequence A.

8. The application of the RNA trans-splicing system based on broken introns as described in any one of claims 1-6 in the construction of RNA sensors, characterized in that, The application includes: constructing a 5' RNA construct and a 3' RNA construct, wherein the 3' EGS is complementary to the target mRNA in the cell to be tested, and the length of the complementary part is greater than the length of the complementary sequence between the 3' EGS and the 5' EGS; the exon is a reporter gene.

9. The application of the RNA trans-splicing system based on broken introns as described in any one of claims 1-6 in constructing biological computing circuits, characterized in that, The application includes: constructing a dual-input logic gate or a multi-input logic gate, transferring it into cells by chemical transformation, and inducing RNA expression using a combination of inducers. The dual-input logic gate is any one of AND gate, NAND gate, implied NOT gate, OR gate, implied gate, or NOR gate, and the multi-input logic gate is an AND gate or a NAND gate.

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