Conditional suicide switch based on CRISPR-Cas conjugated toxin-antitoxin system and its application

Abstract

This invention provides a novel CRISPR-Cas suicide switch system based on TA coupling. By coupling CRISPR-Cas with the TA circuit, a highly efficient and stable suicide switch is achieved. The system includes promoter 1, a CAS protein-coding sequence, promoter 2, a Cascade coding sequence, a CreTA coding sequence, promoter 3, and a guide RNA (gRNA) sequence. The system provided by this invention can exert switching activity in both two-plasmid and single-plasmid systems, exhibiting broad applicability and suicide switch activity suitable for multiple hosts. The suicide switch system provided by this invention is highly efficient, stable, and cross-species applicable, and can be widely used in the fields of genetic engineering and biosafety.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a conditional suicide switch system based on a CRISPR-Cas conjugate toxin-antitoxin system and its application. Background Technology

[0002] In recent years, the CRISPR-Cas system has been widely used in industry, agriculture, medicine, and the environment due to its highly efficient gene-editing capabilities. Advances in genetic engineering and synthetic biology have enabled various genetically modified organisms (GMOs) for open-environment applications. However, the use of GMOs in these applications has raised concerns about the spread of artificially created microorganisms into the natural environment. To address this issue, bioprotective systems have emerged as a method to prevent the unauthorized spread of GMOs and genetic material into ecosystems. However, the genetic stability of bioprotective systems, including suicide switches, remains a challenge that must be addressed. Suicide switches are among the most difficult circuits to maintain because they generate strong selective pressures, leading to a higher probability of escape mutations.

[0003] The CRISPR-Cas system, as a bacterial adaptive immune system, can precisely and efficiently target and destroy bacterial genomes. Therefore, CRISPR-Cas can be used to develop suicide switches, initiating a self-destruct program after the engineered microorganisms have been used to prevent their spread. However, rapidly evolving bacteria can still acquire resistance to CRISPR sterilization through various pathways, such as inactivating Cas nucleases through anti-CRISPR protein Acr (Anti-CRISPR), or disrupting their coding genes through genome rearrangement or transposition events (such as IS elements). Existing suicide switch technologies have shortcomings in stability and cross-species applicability; therefore, a stable and efficient conditional suicide switch system is urgently needed.

[0004] To overcome these challenges, this invention creatively introduces the CreTA system, which acts as an addiction module to prevent the CRISPR effect complex. cascade The loss of the Cascade protein in CreTA can trigger cell dormancy or death. A CreTA-coupled suicide switch not only enables precise and efficient cell suicide under specific conditions but also ensures the stability of the CRISPR-Cas system. This suicide switch system can function effectively in different species and scenarios (such as the animal gut environment) and can be applied to the biocontrol of various engineered bacteria, including live bacterial drugs, fermentation strains, and agricultural inoculants. Summary of the Invention

[0005] This invention utilizes the toxin-antitoxin system CreTA, which effectively maintains the stability of the CRISPR-Cas system in engineered bacteria. A CRISPR-based conditional suicide switch is constructed, and different inducible promoters are used to induce the expression of Cas protein and gRNA, enabling them to bind to and cleave the target genome of the target strain. Simultaneously, the novel toxin-antitoxin system CreTA is used to protect the CRISPR-Cas system, enhancing its stability and preventing environmental pollution risks after the application of engineered probiotics. Therefore, this invention aims to construct a CRISPR-Cas conditional suicide switch based on TA coupling. By constructing the suicide switch using CRISPR-Cas and employing TA-coupled CRISPR-Cas technology, the stability and self-destruction efficiency of the suicide switch are improved, and it can be flexibly applied in multiple species.

[0006] This invention discloses a conditional suicide switch system constructed based on a CRISPR-Cas conjugated toxin-antitoxin system. The system includes a construct 1 and a construct 2. The construct 1 includes the following elements: promoter 1, CAS protein coding sequence, promoter 2, Cascade coding sequence and CreTA coding sequence. The construct 2 includes the following elements: promoter 3 and guide RNA (gRNA) sequence.

[0007] Preferably, the construct 1 includes the following elements in sequence: promoter 1, CAS protein coding sequence, promoter 2, Cascade coding sequence and CreTA coding sequence.

[0008] This invention discloses a conditional suicide switch system constructed based on a CRISPR-Cas conjugated toxin-antitoxin system. The system includes a construct 3, which includes the following elements: promoter 1, a CAS protein coding sequence, promoter 2, a Cascade coding sequence, a CreTA coding sequence, promoter 3, and a guide RNA (gRNA) sequence.

[0009] Preferably, the construct is a plasmid or an expression vector.

[0010] Preferably, the construct 3 comprises the following elements in sequence: promoter 1, CAS protein coding sequence, promoter 2, Cascade coding sequence, CreTA coding sequence, promoter 3, and guide RNA (gRNA) sequence.

[0011] Preferably, the CAS protein is selected from CAS1 protein, CAS2 protein, CAS3 protein, CAS4 protein, CAS5 protein, CAS6 protein, CAS7 protein, CAS8 protein, CAS9 protein and CAS10 protein.

[0012] Preferably, the CAS3 protein sequence is as follows:

[0013] Preferably, promoter 1 and promoter 3 are inductive promoters, and promoter 2 is a constitutive promoter.

[0014] Preferably, promoter 1 is an inductive promoter PcymR, promoter 3 is an inductive promoter Ptet, and promoter 2 is an mPA1 promoter.

[0015] Preferably, the Cascade is derived from Acinetobacter ADP1 strain. csy1-csy2-csy3-csy4 composition.

[0016] This invention discloses the application of the conditional suicide switch system in the fields of microbial gene editing or biosafety prevention.

[0017] Preferably, the microorganisms include bacteria and fungi.

[0018] Preferably, the use includes application to the in vitro animal intestine.

[0019] This invention discloses a kit for microbial gene editing or biosafety control, the kit including the aforementioned conditional suicide switch system.

[0020] This invention discloses the construction of a suicide switch for a dual-plasmid system.

[0021] CRISPR-Cas is coupled with a TA circuit to achieve a highly efficient and stable suicide switch. Figure 1 (A)

[0022] CRISPR-Cas system construction: through reconstruction of Acinetobacter bacillus-derived... A. baylyi The ADP1 CRISPR-Cas system was built in two versions: one with CreTA and the other without, to study the impact of CreTA on the stability of CRISPR-Cas.

[0023] The CIRSPR-Cas system is built on a plasmid with the pSC101 replication origin site, ensuring stable replication of the plasmid in the host cell.

[0024] The plasmid contains a kanamycin resistance gene, providing the host cell with resistance to kanamycin and facilitating the screening of bacteria containing the plasmid.

[0025] This invention uses the constitutive promoter mPA1 to regulate CRISPR-Cas systems. cascade Gene expression is used to maintain the continuous expression of the Cascade protein. (Cascade is derived from Acinetobacter ADP1 strain) csy1-csy2-csy3-csy4 composition) The sequence of mPA1: TTATCCAAAAGAGTATTGACTTAAAGTCTAACCTATAGGATACTTACAAC (SEQ ID NO.2).

[0026] The protein sequence of Csy1 is: MLKIGYITKTGKIMPEPQTDIFINALEESVHFSLKTINSNIEKGGIRLSNQQALDLPYLSARTLLNEDFLIPTDYQANAGKTASIFKVVKAAMISHDISEEDPDFKQQVEQLKPSISSVFNELYQSGTENIDIRMRQLLIPKNGEYVSVSPLTAAGVNYLVNQEVDAVNELRKEKDSDFNRIQTAVFGIGGANPQNVGSLVRSMQRPITVDAPQASEKVRQALAIFYNGFTIRLSRKIKLLNGTKRAKEAIKEWSEILNRQLVITATNTIQNWHDLEIFAPSTNRDVRDQEIIFLNILAKDVLRQASFQKNILEEAEAILPSMQEIERPSFWVHPSLSFVLQGLVDPELRDDHWRIKFSDYLARKITNETFDIEINNETVSISLDSNSEGYIARRLREIIR (SEQ ID NO.3).

[0027] The protein sequence of Csy2 is: MSRYAVIPRIRVQNANMQSNGLLLGGVPLFAANMFAHHLARQLGTTDEGIIYIHHNQQRLGGQAYGRFTPAQRRGAVFIGKKDYSSKNKYALSLQPTASCHLEFSLVIKFSSSRISPEKLTHILKRSRFAGGQIIEFLEITTHAENELEAALKKIKTGFAILDRQDLLIEYQQRQQINRVQAFTQLLALKVDTLRAFFNDQNLSWISATNLGYALLEPLTDQRAGIRQAQDQETTAHAYAEPLTGIVQYFSLGEILRKNTEVEDDSWHNLQKLLWTYHWSQDDIFLLKQNCIHA (SEQ ID NO.4).

[0028] The protein sequence of Csy3 is: MTIFKKLPGSSLSLQRGTVVSDGAFFNLFEDGKQEPLHVMYHGIRGTQNVAGNKEKGAATGNSLAREVTNIQLTESAKLQPNAKLLVKWDFRFIDLSYVLFASASKAGADKTEEYNFRQAFLNFVNRAKESDGLKEVVRRYVRNIVNGRWLWRNRIVAESITIQVTAQDYPETFSFDAL SYNLKSFAEPSEQEQKLAGILLKGITGESTNAALHIEAIVDFGMGGVEVFPSQNYLEDKPKGFSRSLYQLTPKKPDHKANDNQYLGQAALRDQKIGNALRTIDTWYPSFKENDEKPIAIEPNGANLEGQIFYRVGKDKVSAFDILKEIDELDPSSEKGMFLISCLIRGGVYSEGE (SEQ ID NO.5).

[0029] The protein sequence of Csy4 is as follows: MDANYYLDIRVLESSDDTDLKLGHIRNQIYTVIHGAFRKLPAHYALALEMSDKLKAKQEQFEKKHGRSAKPNFDILRIFAEKQDELDELVEAIKGHWKIRDYT VLGVAIAVPTAKISGWKSYRKFRIPTQKAERTKLSHQNEPLRDRRLKTAKGMPFFQVISQSTGQGFTVIIDVQESENAGYGLPDSYGLARKESPFALPVF (SEQ ID NO.6).

[0030] In order to achieve cas3 Gene expression was induced by introducing an inducible promoter P induced by 4-isopropylbenzoic acid (Cumate). cymR Allows activation in the presence of specific chemical inducers. cas3 Gene expression is used to achieve on / off regulation.

[0031] PcymR promoter and regulatory protein sequences: tttacggctagctcagtcctaggtatagtgctagctactagagagCTGTcACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGcctctacaaataattttgtttaaaaagaggagaaatactagatggtaataatgagcccaaaaagaagaacacaagcagaaagagcaatggaaacacaaggaaaactaatagcagcagcactaggagtactaagagaaaaaggatacgcaggattcagaatagcagacgtaccaggagcagcaggagtaagcagaggagcacaaagccaccacttcccaacaaaactagaactactactagcaacattcgaatggctatacgaacaaataacagaaagaagcagagcaagactagcaaaactaaaaccagaggacgacgtaatacaacaaatgctagacgacgcagcagagttcttcctagacgacgacttcagcataagcctagacctaatagtagcagcagaccgcgacccagcactaagagaaggaatacaaagaacagtagaaagaaacagattcgtagtagaggacatgtggctaggagtgctagtaagcagaggactaagcagagacgacgcagaggacatactatggctaatattcaacagcgtaagaggactagcagtaagaagcctatggcaaaaagacaaagaaagattcgaaagagtaagaaacagcacactagaaatagcaagagaaagatacgcaaaattcaaaagataataaCTCGGTACCAAATTCCAGAAAAGAGGCCTCCCGAAAGGGGGGCCTTTTTTCGTTTTGGTCCtactagaggttatgagtcaggaaaaaaggcgacagagtaatctgtcgccttttttctttgcttgctttaaatcataaaaaatttatttgctttgtggcggataacaattataatagattcaacaaacagacaatctggtctgtttgtatt(SEQ IDNO.7)。

[0032] The CreTA disclosed in this invention is a toxin-antitoxin (TA) system that is structurally and functionally highly coupled to CRISPR. The toxin CreT is an RNA or protein-based toxin, while the antitoxin CreA is a small RNA molecule with a crRNA-like structure. It works by binding with… creT Partial sequence matching between gene promoters guides CRISPR Cascade to repress CreT expression, stably inhibiting the transcription of the toxin CreT. Once the Cascade component is disrupted, the inhibition of the toxin CreT is released, and the CreT toxin can then be expressed normally, thereby inhibiting / killing the host bacteria and protecting them. cascade Genetic stability of gene elements ( Figure 1 (A)

[0033] CreTA sequences: AAGAGAAAGTTTTAATGAAAGAATACAGTCCTAAAGCGACATCTAATGTCGTTTTAAATCTATTTTTTAGCTTGTGTTTACTTAAAAAATAGTTGCAAGCGGATGAAGCAGTTTCTGTATAATAGCTATTGTTCGCTACTGCATAAGTAGTTGAGAAAGAGACTAGATTCTTGTTTAAGAACTATCTCGTTGACCATCGCTTTAGATGGATTACTTTTTGTTACATCTAAAATTTCATTGAATAACAACAATATAAATTCAAATAAAAAATAATGCCCGAGACTAGATTGTTGTTTACTTGATATAAAAATTTAACTAGTCATAATAAACTAGAACTGAGCAGCACAAAGCATCTCAACTCTTTTGTGCTGCTCTTCAAGTTAACTCGTATCAAAAGGAGCTCACTCATGAGTCATCTTATCACAGAACATGTAGATCCATTCTCAAATCAAGAAAAATCATCACCTGATTTTTCACTGAGTATGTCAGCAAAACTTCTAAAGTTATCTCCTAAAGTATTTATTGAACTCTTATACAGGAATAAATTTATTTTTAAACGAAGTAAGCAAACCGCATGGGAGTCATATCAAAATATTGTAGATAGAGAAATCTTGTGCCATCGAGTAATAAACATAAAGCACTCTACAGGCATAGTAGTTCCACATTTACAAGTTAGAGTCACTTTAAAAGGTCTAACATATTTTAAAAATAGATTTTGCTCTTAAATCTACGAAGATATTATTGCGACATTTGATGTCGCAATACTACTTAATATTTATC (SEQ ID NO.8).

[0034] Preferably, the CreA sequence is: gttcgctactgcataagtagttgagaaagagactagattcttgtttaagaactatctcgttgaccatcgctttagatggattactt (SEQ ID NO.9).

[0035] The protein sequence of CreT is: MSSTKHLNSFVLLFKLTRIKRSSLMSHLITEHVDPFSNQEKSSPDFSLSMSAKLLKLSPKVFIELLYRNKFIFKRSKQTAWESYQNIVDREILCHRVINIKHSTGIVVPHLQVRVTLKGLTYFKNRFCS (SEQ ID NO. 10).

[0036] To test the bactericidal activity of the CRISPR-Cas system in Escherichia coli Nissle 1917 (EcN), a targeting gRNA was constructed based on E. coli EcN, targeting essential gene sites on the genome.

[0037] The present invention discloses anhydrous tetracycline-induced Ptet promoter regulation of guide RNA (gRNA), p15A replication initiation site, and chloramphenicol resistance.

[0038] Sequences of the Ptet promoter and its regulatory proteins: (SEQ ID NO.11).

[0039] This invention discloses the experimental verification of the bactericidal circuit: gRNA with the ability to target the target gene and a control plasmid without the ability to target the gene were transformed into the ATTACH (PcymR-Cas3_mPA1-Cascade) system, respectively. When the gRNA is present, it binds to the Cascade in the PcymR-Cas3_mPA1-Cascade system, guiding the Cas3 nuclease to cleave the target site, initiating DNA degradation, thereby achieving the bactericidal purpose. Clones containing gRNA exhibited cleavage activity and the strains could not grow, while the control strains without gRNA grew normally.

[0040] This invention discloses the verification of the CreTA circuit: the CreTA circuit is verified by blocking the normal functioning of Cascade with anti-CIRSPR protein (Acr).

[0041] This invention further designs and constructs an ultra-strict suicide switch: due to the leakage expression of the gRNA promoter, bacteria are killed under non-inducible conditions. To achieve a strict switch effect where colonies grow normally under non-inducible conditions but are killed under induced conditions, the promoter and 5'UTR region of the gRNA are mutated to change the regulatory region, reduce background expression, and achieve a more stringent switch effect. Figure 4 ).

[0042] This invention further screens and tests the bactericidal activity of different RNA targets. To test the bactericidal activity of different RNA targets, [the invention targets specific RNA targets]. frr, thiL, recA, groL, groS, rrsH Genes were designed and corresponding gRNA targets were constructed and transformed into CRISPR-Cas system strains.

[0043] This invention discloses the advantages of a suicide switch coupled with a current transformer (TA).

[0044] This invention discloses a cross-species single-plasmid system suicide switch: developing a plug-and-play suicide switch toolkit applicable to different species.

[0045] After testing the switching activity of the dual-plasmid system and evaluating the advantages of the CreTA system, this invention successfully integrated guide RNA (gRNA) and the CRISPR-Cas system into a single plasmid to improve ease of use, thus constructing a single-plasmid switching system.

[0046] This invention discloses an animal intestinal environment test: testing the effectiveness of the suicide switch in the animal intestinal environment to evaluate its practical application potential. Attached Figure Description

[0047] Figure 1 Design of a suicide switch based on a CRISPR-Cas conjugated toxin-antitoxin (TA) system. A represents a constructed CRISPR-Cas suicide switch system combined with TA; B is a functional diagram of the TA loop verified using Acr-IF25; C demonstrates the TA loop's conversion efficiency by evaluating conversion rates with and without Acr through plate counting.

[0048] Figure 2 The sterilization circuit was validated by conversion counting.

[0049] Figure 3 Designing and constructing an extremely rigorous suicide switch, among which... Figure 3 A is a rigorous switch for screening promoter and 5'UTR mutation libraries; Figure 3 B represents the screening of multiple gene targets.

[0050] Figure 4 Advantages of the suicide switch coupled with TA.

[0051] Figure 5 The escape rates of the suicide switch for + / - TA in single plasmid systems and different species. A represents the integration of CRSIPR-Cas and gRNA into a single plasmid system, which can be used in multiple species; B represents the escape rate assay method; C represents the escape rate of + / - TA suicide switch in *E. coli* Nissle1917 (EcN); D represents the escape rate of + / - TA suicide switch in *E. coli* MG1655; E represents the escape rate of + / - TA suicide switch in *Salmonella* (Se); and F represents the escape rate of + / - TA suicide switch in *Klebsiella pneumoniae*.

[0052] Figure 6 The efficiency of the suicide switch in the animal intestinal environment. Specifically: A) the suicide switch efficiency of EcN-ATTACH-V2 in the rat intestinal environment; B) the number of engineered bacteria surviving in rat feces under different conditions; and C) the suicide efficiency of EcN-ATTACH-V2 in the animal intestine with + / -TA. Detailed Implementation

[0053] Example 1: Development and Validation of a Suicide Switch Based on a CRISPR-Cas Conjugated Toxin-Antidote (TA) System 1.1 A highly efficient and stable suicide switch was designed and developed by integrating toxin-antitoxin (TA) with the CRISPR-Cas system. This was achieved through the study of strains... A. baylyi The ADP1 CRISPR-Cas system was redesigned to construct a TA-coupled CRISPR-Cas suicide switch system (Figure 1A). Cas3 protein expression is regulated by the 4-isopropylbenzoic acid (Cumate)-induced promoter PcymR, while guide RNA (gRNA) expression is controlled by the ahydrotetracycline (aTc)-induced promoter Ptet. Constitutively expressed Cascade, guided by CreA, inhibits the expression of the toxin CreT (Figure 1A). These elements together constitute a two-input suicide switch system named ATTACH. When Cascade mutates or recombines, or is disrupted by a moving element, the toxin is released, leading to cell dormancy / death and triggering the self-destruct circuitry. This design enhances the stability and efficiency of the suicide switch.

[0054] 1.2 Experimental Verification of the Sterilization Circuit gRNAs capable of targeting specific genes and control plasmids without targeting capabilities were transformed into the ATTACH (PcymR-Cas3_mPA1-Cascade) system, respectively. In the presence of gRNA, it binds to the Cascade plasmid in the ATTACH system, guiding the Cas3 nuclease to cleave the target site, initiating genomic DNA degradation, thereby achieving bactericidal activity. The group containing gRNA exhibited cleavage activity, and the strains could not grow, while the control strains without gRNA grew normally. This result clearly demonstrates that the ATTACH system exhibits significant bactericidal activity under the action of gRNA. Figure 2 ).

[0055] 1.3 Verification of CreTA loops The CreTA circuit was validated by blocking the normal functioning of Cascade using the anti-CIRSPR protein AcrIF25.

[0056] AcrIF25 expression is controlled by the promoter Ptet, with plasmid P15A as the replication origin and chloramphenicol resistance. This plasmid was designed to test the CRISPR-Cas line-coupled CreTA circuit.

[0057] The constructed AcrIF25 plasmid and the empty plasmid control were transformed into a CRISPR-Cas system containing CreTA, plated and counted to calculate the transformation efficiency.

[0058] Experimental results showed that colonies grew normally in the absence of Acr, indicating that the CRISPR-Cas system remains stable without an inducer. However, colonies failed to grow normally in the presence of Acr, suggesting that Acr disrupts the Cascade gene in the CRISPR-Cas system, relieving the inhibition of CreT and allowing CreT expression to achieve a bactericidal effect. Figure 1 (C)

[0059] Figure 1 The diagram below shows the verification of the TA circuit using AcrIF25.

[0060] Figure 1 In C-cell transformation plate colony counting: To validate the TA loop, AcrIF25 was used to disrupt the cascade. Transformation efficiency was assessed by plate counting, and the transformation rates with and without Acr were compared, demonstrating the successful construction of the TA loop.

[0061] Figure 2 Transformation experiments demonstrated that the CRISPR-Cas system possesses effective cleavage activity.

[0062] 1.4 Design and Construction of an Ultra-Strict Suicide Switch Due to promoter leakage expression of gRNA, under non-inducible conditions, the RNA plasmid directly exerts bactericidal activity after transformation into cells. Figure 2 To achieve a precise on / off effect where colonies grow normally under non-induced conditions but are killed under induced conditions, a targeted gene is used. groL The gRNA (gRNA-groL) is modified by mutating the promoter and 5'UTR region of the gRNA, altering the regulatory region, reducing background expression, and achieving a more precise on / off effect. Figure 3 A).

[0063] A mutant library containing the gRNA promoter and 5'UTR was transformed into an EcN strain of the ATTACH system containing CRISPR-Cas for library construction and screening. Single colonies from the transformed plates were spotted onto resistant plates without an inducer and resistant plates with an inducer (Cumate 0.3 mM + aTc 400 ng / mL). After incubation at 37°C for 12 h, the growth differences between the resistant and inducible plates were observed. Strains that grew normally on resistant plates without an inducer but did not grow on resistant plates with an inducer were identified as having switch activity.

[0064] (1) Preparation of LB solid induction medium plates: A specific concentration of antibiotic, Cumate (0.3 mM), and aTc (400 ng / mL) were added to LB solid medium to prepare solid medium plates containing inducing agents. The prepared medium plates were then cooled at 4°C for subsequent experiments.

[0065] (2) Colony plating and streaking: Single colonies were selected from the electroporated medium plates and first streaked on resistant LB plates without the addition of an inducer to assess colony growth. Then, the streaking operation was repeated on resistant LB plates containing the inducer to detect the effect of the inducer on colony growth.

[0066] (3) Setup of experimental and control groups: For the experimental group, mutant monoclonal colonies were selected and spotted onto plates with + / - inducers, and the operation was repeated for verification. For the control group, strains lacking switching activity in the same system were selected to verify that the switching activity observed in the experimental group was not caused by the inducer.

[0067] (4) Cultivation and observation: Place the streaked plates in a 37°C incubator for 8 to 12 hours. After cultivation, compare and observe the growth status of the mycelium on the two plates.

[0068] The results showed that this invention successfully screened strains that grew normally on resistant plates without an inducer, but did not grow on resistant plates with an inducer, indicating that these strains possess the desired switching activity. A system with switching effect was constructed, named ATTACH-V1, with the preferred sequence as follows: Figure 3 As shown in Figure A.

[0069] 1.5 Screening and testing RNA targets of different genes This invention screened for six essential gene targets in Escherichia coli (E. coli). frr, thiL, recA, groL, groS and rrsH ).

[0070] Based on the preferred 5'UTR sequence obtained through gRNA-groL mutation screening, the target sequence was replaced to screen six essential genes with different physiological functions to find the best target (Figure 3B). The targets of five of these genes can achieve a strict on / off effect.

[0071] To test the bactericidal activity of different RNA targets, targeting frr, thiL, recA, groL, groS and rrsH Genes were designed and corresponding gRNA targets were constructed and transformed into CRISPR-Cas strains. Single colonies were selected from antibiotic-resistant plates and inoculated into LB broth containing antibiotics to obtain sufficient cell density. After 12 hours of culture, the seed culture was diluted 300-fold and transferred to fresh LB broth. One group was treated with inducers (Cumate 0.2 mM and aTc 200 ng / mL), while the other group was treated without inducers, to compare the suicide switch effect of different target gRNAs. After 8 hours of culture, the optical density (OD value) of each culture medium was measured to assess cell growth.

[0072] (1) The experimental results showed that, without the addition of an inducer, the colony biomass of all target sites was comparable to that of the control, indicating that the colony growth was normal.

[0073] (2) In the control group, there was no significant difference in OD values ​​between gRNAs that did not target any gene under induced and non-induced conditions, indicating that the CRISPR-Cas system does not affect cell growth in the absence of specific gRNA targeting.

[0074] (3) Under the condition of induction by an inducer, for f rr, thiL, recA, groL and GroS The gRNAs of five genes have a precise bactericidal on / off switch effect. Figure 3 (B)

[0075] 1.6 Advantages of the suicide switch coupled with TA Using the screened targets groLThe guide RNA (gRNA-groL) plasmid of the gene was transformed into ATTACH strains containing + / - CreTA. The selected single colonies were inoculated into liquid LB medium containing antibiotics and cultured overnight (12 hours). The cultured seed culture was diluted 300-fold and transferred in equal volumes to new liquid LB medium containing (cumate 0.2 mM and aTc 200 ng / mL) or without an inducer.

[0076] Three hours later, an appropriate amount of bacterial suspension was diluted and spread onto resistant plates containing (Cumate 0.3 mM + aTc 400 ng / mL) inducer and plates without inducer, and incubated at 37°C for 12 hours. After incubation, single colonies were counted, and CFU / mL (colony forming units / mL) was calculated. The results are as follows: Figure 4 As shown.

[0077] (1) The results showed that under induction conditions, the CFU values ​​of colonies containing CreTA system and those without CreTA system were significantly lower than those of the non-inducible group. This phenomenon indicates that under the guidance of gRNA, the CRISPR-Cas system can effectively enable engineered bacteria to commit suicide under induction conditions and has strict on / off activity.

[0078] (2) Further analysis showed that, under induction conditions, the biomass of the system containing CreTA was significantly lower than that of the system without CreTA, indicating that the introduction of the CreTA system significantly enhanced the suicide switch effect of the CRISPR-Cas system. Figure 4 ).

[0079] This invention designs and constructs a novel suicide switch, which is more effective and efficient based on the CRISPR-Cas conjugate toxin-antitoxin (TA) system.

[0080] Example 2: Development of a single-plasmid suicide switch system and system escape rate testing Develop a plug-and-play kill switch toolkit suitable for different species.

[0081] After testing the switching activity of the dual-plasmid system and evaluating the advantages of the CreTA system, to improve ease of use and expand its application scenarios, this invention integrates the gRNA and CRISPR-Cas from the dual-plasmid ATTACH system into a single plasmid, constructing the single-plasmid switching system ATTACH-V2. The guide RNA (gRNA) and CRISPR-Cas from the dual-plasmid system were separately PCR-PCRed and assembled using Gibson technology to form a single-plasmid system. Based on this, NotI digestion was performed to insert the essential gene infA as a selection marker in the absence of antibiotics, for subsequent testing in antibiotic-free environments in animal experiments.

[0082] The ATTACH-V2 system is constructed as follows: PSC101 replication origin site, chloramphenicol resistance. gRNA expression is regulated by the tetracycline-inducible promoter Ptet, while... cas3 Gene expression is controlled by the inducible promoter PcymR, while cascade gene expression is regulated by the constitutive promoter (Pcon) mPA1. Figure 5 Furthermore, the infA gene was knocked out in the genome of *Escherichia coli* Nissle 1917 (EcN) and reintroduced into the ATTACH-V2 system as an essential gene defect selection marker. By transferring the single-plasmid switch system into *EcN*, MG1655, *Klebsiella pneumoniae* (KP), and *Salmonella enterica* (Se), plug-and-play functionality of the single-plasmid switch system in multiple species was achieved.

[0083] Cross-species suicide switch escape rate comparison: Comparing the suicide switch escape rates of different species, and the effect of + / -TA.

[0084] (1) Screening of engineered strains with suicide switch effect: Engineered strains with switch effect were screened through platelet analysis and liquid culture tests. This included two groups of strains: one containing the CreTA system and the other without the CreTA sealing system. Subsequently, single colonies were picked from these strains and inoculated into liquid LB medium containing the corresponding antibiotics for culture. The bacteria were cultured in a shaker at 220 rpm and 37°C for 12 hours until they reached the plateau phase.

[0085] (2) Dilution and transfer of seed culture: The seed culture cultured overnight was diluted 300 times and transferred in equal amounts to resistant liquid LB medium containing inducer (cumate 0.2mM, aTc 200ng / mL) and without inducer, in order to evaluate the effect of inducer on strain growth and switching activity.

[0086] (3) Sampling and plating: Starting from 0 hours after transfer, samples were taken every hour, and the bacterial solutions from different time periods were serially diluted and then plating. The induced group strains were plating onto resistant plates containing inducers, and the non-induced group strains were plating onto resistant plates without inducers.

[0087] (4) Culture and Colony Counting: Place the plated plates in a 37°C incubator and invert them for 12 hours. After culture, count the colonies to assess the system on / off effect and escape frequency. Escape frequency = (number of colonies in the induced group / number of colonies in the non-induced group) × 100% Figure 5 (B)

[0088] The results showed that the single plasmid suicide switch ATTACH-V2 in Escherichia coli (EcN) Figure 5 (C), MG1655 ( Figure 5 (D), Salmonella (Se) Figure 5 (E) and Klebsiella pneumoniae (Kpn) Figure 5 F) All strains exhibit switching activity, and strains without the CreTA system have an escape rate between 10%. -2 Up to 10 -5 The escape rate of strains with the CreTA system is between 10 and 10%. -6 Up to 10 -8 Between different species, the escape rate of the suicide switch loaded with CreTA was lower than that of the group without CreTA. Figure 5 (CF). Therefore, by integrating CRISPR-Cas with Ptet-RNA in the dual-plasmid ATTACH system, a single-plasmid suicide switch system, ATTACH-V2, was developed. This system supports cross-species function, enabling plug-and-play use with different species such as Escherichia coli probiotics Nissle1917 (EcN), MG1655, Salmonella (Se), and Klebsiella pneumoniae (Kp). This system has broad applicability and possesses suicide switch activity suitable for multiple hosts. Therefore, this suicide switch system can be applied to the biocontrol of various engineered bacteria, including live bacterial drugs, fermentation strains, and agricultural inoculants.

[0089] Example 3: In vivo animal testing of the single-plasmid system ATTACH-V2 suicide switch The efficiency and stability of the suicide switch were tested in an animal gut environment to assess its potential for practical applications.

[0090] This study simulates the process of inducing the self-destruction and clearance of engineered probiotics after administration into the animal gut, evaluating and comparing the efficiency and stability of self-destruction switch systems containing CreTA and those without CreTA (+ / -TA). For ease of comparison, a mixed bacterial design was introduced, where strains with +TA and -TA were mixed at a 1:1 biomass ratio. After treatment with an inducer in animals, the proportion of remaining bacteria in feces was assessed to determine which strain exhibited better self-destruction switch efficacy. Figure 6 (A)

[0091] First, mice that had been adapted for one week were given a mixed antibiotic treatment in their drinking water for 8 days to obtain germ-free mice.

[0092] Antibiotic treatment conditions: Add the following antibiotic mixture to the drinking water of mice: 1 g / L ampicillin, 1 g / L neomycin, 0.5 g / L vancomycin, 0.5 g / L metronidazole, 0.5 g / L gentamicin, and 2.5 g / L sucralose.

[0093] The mice were then divided into four groups of six: Group 1 (Control) - No inducer (Control Inducer) was administered the Control strain and followed by normal drinking water; Group 2 (Control) - with inducer (Control + Inducer), the Control strain was given and subsequently supplemented with inducer in drinking water; Group 3+ / TA mixed bacteria - without inducer, administered mixed bacteria and followed by normal drinking water; Group 4+ / -TA mixed bacteria - with inducer (Mixture + Inducer), the mixed bacteria were given and the inducer was added to the drinking water afterwards.

[0094] After switching to normal drinking water, the mice were given a two-day interval to rule out the effects of antibiotics. Then, for seven consecutive days, the mice were given 10^9 CFU of control strains (without gRNA) from groups 1 and 2, and 10^9 CFU of mixed strains from groups 3 and 4, by gavage.

[0095] Subsequently, the inducing agent dose (Cumate, 30 mM, aTc: 100 ug / ml) was added to the drinking water of mice in groups 2 and 4, while mice in groups 1 and 3 continued to drink water normally. Fecal samples were collected from mice at 0, 24, 48, 72, 120, and 168 hours.

[0096] Collected fecal samples were ground, diluted, and spread onto antibiotic plates, and the number of colonies was counted. Results showed that the bacterial load in the feces of mice given the Control strain remained unchanged regardless of whether they received an inducer in their drinking water. Mice given a mixed bacterial strain and normal drinking water (Mixture-Inducer group) showed no significant difference compared to the Control group. However, in mice given a mixed bacterial strain and an added inducer in their drinking water (Mixture + Inducer group), the number of viable bacteria in the feces began to decrease after 24 hours and was significantly lower than that of mice with normal drinking water (Mixture-Inducer group) after 120 hours, decreasing by four orders of magnitude. This indicates that after the control system takes effect in the mouse intestine, it can be effectively cleared by the inducer. Figure 6 (B)

[0097] High-throughput sequencing was used to determine the proportions of +TA and -TA strains in the feces of mice treated with the mixed bacteria. Statistical results showed that the proportions of +TA and -TA bacteria remained essentially unchanged in the feces of mice not treated with the inducer. However, in mice treated with the inducer, the proportions of +TA and -TA bacteria in the feces changed significantly after 24 hours, with the proportion of CreTA-protected suicide-switching strains (+TA bacteria) dropping sharply to below 0.2%. This result indicates that +TA bacteria were significantly eliminated under the influence of the inducer, while the remaining colonies were predominantly -TA bacteria not protected by CreTA. This fully demonstrates that CreTA's protection of the containment system can improve containment efficiency and effectively prevent strain escape. Figure 6 (C). Therefore, this suicide switch system works effectively in the complex environment of the animal gut and can be applied to the biocontrol of live bacterial drugs and various bacterial agents.

Claims

1. A conditional suicide switch system based on a CRISPR-Cas conjugated toxin-antitoxin system, the system comprising construct 1 and construct 2, characterized in that, The construct 1 includes the following elements: promoter 1, CAS protein coding sequence, promoter 2, Cascade coding sequence, and CreTA coding sequence; the construct 2 includes the following elements: promoter 3 and guide RNA (gRNA) sequence. Promoter 1 and promoter 3 are inductive promoters, and promoter 2 is a constitutive promoter.

2. The conditional suicide switch system according to claim 1, characterized in that, The construct 1 includes the following components in sequence: promoter 1, CAS protein coding sequence, promoter 2, Cascade coding sequence and CreTA coding sequence.

3. A conditional suicide switch system based on a CRISPR-Cas conjugated toxin-antitoxin system, the system comprising a construct 3, characterized in that, The construct 3 includes the following elements: promoter 1, CAS protein-coding sequence, promoter 2, Cascade coding sequence, CreTA coding sequence, promoter 3, and guide RNA (gRNA) sequence; Promoter 1 and promoter 3 are inductive promoters, and promoter 2 is a constitutive promoter.

4. The conditional suicide switch system according to claim 3, characterized in that, The system includes a construct 3, characterized in that the construct 3 sequentially includes the following elements: promoter 1, a CAS protein coding sequence, promoter 2, a Cascade coding sequence, a CreTA coding sequence, promoter 3, and a guide RNA (gRNA) sequence; Promoter 1 and promoter 3 are inductive promoters, and promoter 2 is a constitutive promoter.

5. The conditional suicide switch system according to any one of claims 1-4, characterized in that, The CAS proteins are selected from CAS1, CAS2, CAS3, CAS4, CAS5, CAS6, CAS7, CAS8, CAS9 and CAS10 proteins.

6. The conditional suicide switch system according to any one of claims 1-4, characterized in that, Promoter 1 is the inducible promoter PcymR, promoter 3 is the Ptet promoter, and promoter 2 is the mPA1 promoter.

7. The conditional suicide switch system according to claim 1, characterized in that, The Cascade consists of csy1-csy2-csy3-csy4 derived from the A. baylyiADP1 strain.

8. The use of the conditional suicide switch system according to any one of claims 1-7 in the field of microbial gene editing or biosafety prevention; wherein the microorganism is bacteria.

9. A kit for microbial gene editing or biosafety control, characterized in that, The kit includes the conditional suicide switch system according to any one of claims 1-7; the microorganism is bacteria.

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