A reversibly regulated complex of an IB-type CRISPR-Cas system based on ADP-ribosylation modification and its applications

Abstract

This invention relates to a reversible regulatory complex of a type I-B CRISPR-Cas system based on ADP-ribosylation modification and its applications, belonging to the field of gene editing technology. In the type I-B CRISPR-Cas system of this invention, the anti-CRISPR protein AcrIB8 and its reverse regulator ARH protein, AcrIB8 possesses ART enzyme activity and can specifically modify the Cas7b subunit of the Cascade complex, thereby blocking the function of the complex; the ARH protein can reverse this modification and restore the activity of the CRISPR system, thus constructing a reversible "modification-demodification" regulatory switch. This invention provides a regulatory tool for type I-B CRISPR-Cas gene editing for the first time, and further solves the problems of irreversibility and imprecise regulation of existing Acr tools, providing core components and methods for safe and controllable gene drug development and precise gene editing.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to a reversible regulatory complex of an IB-type CRISPR-Cas system based on ADP-ribosylation modification and its application. Background Technology

[0002] CRISPR-Cas gene editing technology has been widely applied in gene therapy, genetic disease treatment, and tumor immunotherapy. However, off-target effects and uncontrollable activity remain core bottlenecks in clinical translation. Natural anti-CRISPR (Acr) proteins can serve as endogenous regulatory switches for the CRISPR system; however, most Acr proteins rely on protein-protein interactions to achieve stoichiometric inhibition, which suffers from low efficiency, irreversibility, and poor specificity. In recent years, a few enzymatic Acr proteins have been found to achieve catalytic inhibition through post-translational modifications such as ADP-ribosylation, exhibiting high efficiency and durability. However, these covalent modifications lack corresponding demodification reversal mechanisms, making it difficult to achieve precise "on-off" regulation of CRISPR activity, which significantly limits their application in safe gene editing. Type I-B CRISPR-Cas systems offer advantages such as long fragment deletion, low off-target effects, and large-capacity delivery, making them more suitable for gene therapy and synthetic biology applications. Therefore, developing reversible regulatory tools for these systems has high clinical translational value. Within the current technological scope, there is still a lack of tools for the reversible regulation of enzymatic inhibition-deinhibition of the type I-B CRISPR-Cas system. At the same time, no anti-anti-CRISPR (Aacr) factor that can specifically reverse AcrIB8 modification has been found. Summary of the Invention

[0003] To address the aforementioned issues, this invention co-expresses the IB-type CRISPR system, AcrIB8, and ARH in cells or organisms, achieving reversible switching of editing activity "off" to "on" through time- and dose-controlled methods, thus realizing precise regulation of gene editing. In this invention, AcrIB8 is first used to inhibit non-specific editing, significantly reducing the off-target probability, and then ARH is used to restore targeted editing as needed. In terms of application, AcrIB8-ARH can be incorporated into gene therapy vectors as a "safety braking module," enabling controllable on / off switching of editing activity and serving as a safety switch for gene therapy.

[0004] The first objective of this invention is to provide a reversibly modulated complex for a CRISPR-Cas system, comprising:

[0005] Anti-CRISPR protein (Acr), the amino acid sequence of which is shown in SEQ ID NO.1;

[0006] The anti-CRISPR protein reverse regulator (Aacr / ARH protein) has an amino acid sequence that is one of the sequences shown in SEQ ID NO. 8-10. ARHs from different sources exhibit significant functional differences, with SpARH, shown in SEQ ID NO. 10, showing the strongest reversal activity. Therefore, we not only discovered the ARH family of functional proteins, but also identified the optimal combination with AcrIB8 from multiple candidate molecules, providing the best components for the subsequent development of efficient and precise CRISPR regulatory tools.

[0007] A second objective of this invention is to provide a nucleic acid sequence encoding the reversible regulatory complex of the CRISPR-Cas system.

[0008] A third objective of this invention is to provide a recombinant plasmid containing the said nucleic acid sequence.

[0009] A fourth objective of this invention is to provide recombinant cells containing the CRISPR-Cas system reversible regulatory complex or the nucleic acid sequence.

[0010] Furthermore, the host cell of the recombinant cell is a non-plant cell, preferably a prokaryotic cell, such as Escherichia coli.

[0011] A fifth objective of this invention is to provide the application of the CRISPR-Cas system reversible regulatory complex, nucleic acid sequence, recombinant plasmid, or recombinant cell in the preparation of gene editing products or gene drugs.

[0012] Furthermore, the gene editing product is used for reversible regulation of gene editing.

[0013] A sixth object of the present invention is to provide a gene editing system comprising:

[0014] Cascade complex, wherein the Cascade complex contains a Cas7b subunit;

[0015] Cas protein (used to recognize and cleave target nucleic acids);

[0016] The CRISPR-Cas system can reversibly regulate complexes.

[0017] Furthermore, the Cascade complex contains Cas5b subunit, Cas6b subunit, Cas7b subunit, Cas8b subunit, Cas11b subunit, and crRNA.

[0018] Furthermore, the amino acid sequences of the Cas5b subunit, Cas6b subunit, Cas7b subunit, Cas8b subunit, and Cas11b subunit are shown in SEQ ID NO.2-6, respectively.

[0019] Furthermore, the crRNA is designed to target nucleic acids and is used to guide the Cascade complex to locate the target nucleic acids.

[0020] Furthermore, the Cas protein includes, but is not limited to, the Cas3b protein (used for reversible regulation of the IB-type CRISPR-Cas system).

[0021] Furthermore, the amino acid sequence encoding the Cas3b protein is shown in SEQ ID NO.7.

[0022] Furthermore, in the gene editing system, the molar ratio of Cas5b subunit, Cas6b subunit, Cas7b subunit, Cas8b subunit, Cas11b subunit, and crRNA can be 1:1:(5-8):1:3:(2-4), the molar ratio of Cascade complex to Acr is 1:(0.01-10), and the molar ratio of Cascade complex to ARH is 1:(0.01-10). The ratios used in the following examples are: the molar ratio of Cas5b subunit, Cas6b subunit, Cas7b subunit, Cas8b subunit, Cas11b subunit, and crRNA is 1:1:7:1:3:3, the molar ratio of Cascade complex to Acr is 1:0.1, and the molar ratio of Cascade complex to ARH is 1:0.1.

[0023] Furthermore, the temperature range for reversible regulation is 4-42℃, with the most preferred range being 21-25℃. As an enzyme-catalyzed reaction effector, the activity of ARH protein is usually temperature-regulated, and it can maintain high catalytic efficiency within a mild physiological temperature range (such as 21-25℃). The effective temperature range for ARH protein to restore the DNA-binding activity of the CasCade complex can be controlled at around 21-25℃. In this invention, a time-dependent band enhancement was observed under an incubation condition of 23℃ for 8 hours.

[0024] The regulatory mechanism of the gene editing system of this invention is as follows:

[0025] (1) Turn off CRISPR: AcrIB8 → Modifies Cas7b → Blocks Cascade assembly → Inhibits target DNA binding and cleavage;

[0026] (2) Activate CRISPR:ARH → Remove ADP-ribosylation of Cas7b → Restore Cascade assembly and DNA binding → Rebuild immune / editing activity.

[0027] A seventh object of the present invention is to provide recombinant cells containing the gene editing system described above (host cell limitation as above).

[0028] An eighth object of the present invention is to provide the application of the gene editing system in the preparation of gene editing products or gene drugs.

[0029] A ninth object of the present invention is to provide the use of any of the sequences shown in SEQ ID NO. 8-10 or sequences with a sequence similarity of 90% or more (e.g., 95% or more, especially 97%, 98% or more or 99% or more) in the preparation of a reverse regulator of the anti-CRISPR protein shown in SEQ ID NO. 1.

[0030] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0031] 1. Novel “enzyme-active” inhibition mechanism: This invention reveals for the first time that AcrIB8 inhibits the CRISPR-Cas system through ADP-ribosylation, a post-translational modification, which is different from traditional protein-protein interactions and provides a novel molecular strategy for gene editing regulation.

[0032] 2. High efficiency and long duration: As an enzyme activity inhibitor, it only requires a small amount of catalyst to achieve permanent inactivation modification of the CRISPR system, with high inhibition efficiency and long duration.

[0033] 3. First achievement of reversible regulation: This invention is the first to screen and verify the ARH protein (Aacr) that can specifically reverse AcrIB8 modification, and successfully constructs the "AcrIB8-ARH" reversible regulatory pair, realizing closed-loop control from "inhibition" to "recovery", filling the technological gap in this field.

[0034] 4. High targeting specificity: AcrIB8 specifically modifies Cas7b, while ARH can specifically remove this modification. Together, they form a precise molecular switch, providing a core component for developing next-generation gene drugs with "off" and "reboot" functions, which is expected to greatly improve the safety of gene editing. Attached Figure Description

[0035] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0036] Figure 1 The images show the detection results of ADP-ribosylation modification of the Cascade complex and Cas3b by AcrIB8. The left image shows the results of Western blot detection, with molecular weight in kDa, and the right image shows the SDS-PAGE Coomassie Brilliant Blue stained gel image.

[0037] Figure 2 The images show the results of reversing the AcrIB8 modification of the Cascade complex by ARH proteins from three sources. (A) shows the reduction of the ADP-ribosylation modification signal, and (B) shows the Coomassie Brilliant Blue staining of the corresponding samples.

[0038] Figure 3 The diagram shows the results of the addition of three different species of ARH restoring Cascade's binding to DNA to varying degrees.

[0039] Figure 4 This is a diagram showing the inactivation of the IB-type CRISPR system caused by the ADP-ribosyltransferase activity of AcrIB8.

[0040] Figure 5 This is a graph showing the results of three ARH proteins from different sources reversing the inhibitory activity of AcrIB8 to varying degrees in vivo. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0042] I. The solution involved in this invention is as follows:

[0043] In terms of CRISPR-Cas regulation, the existing technologies have the following defects and shortcomings: (1) The inhibition mode is single and irreversible: Most of the existing Acr are stoichiometric binding, which requires a large amount of expression to effectively inhibit them, and it is difficult to restore the system activity after inhibition, lacking reversible regulation ability; (2) There is a lack of efficient enzyme active inhibitors: There are very few reports on Acr (enzyme active Acr) that use post-translational modification for "catalytic" inhibition, and its mechanism of action and advantages have not been fully explored; (3) There is a lack of effective reversal tools: For covalent modification mediated by enzyme active Acr, there are no anti-anti-CRISPR (Aacr) reverse regulators that can specifically reverse the modification and restore CRISPR activity.

[0044] In view of the above-mentioned shortcomings of the prior art, the present invention provides the following solutions: (1) a novel Acr protein with ADP-ribosyltransferase (ART) activity is provided, which achieves efficient and durable CRISPR inhibition through covalent modification; (2) an ADP-ribosylhydrolase (ARH) protein that can specifically reverse the above-mentioned Acr protein modification effect is provided, forming a reversible regulatory pair; (3) the above-mentioned Acr-ARH composition is provided for the application in the preparation of precise and reversible CRISPR gene editing regulatory tools.

[0045] This invention achieves reversible regulation (tool value): it demonstrates that "AcrIB8 inhibition-ARH deinhibition" forms a complete molecular switch. This provides direct evidence for developing this protein pair into a controllable gene editing tool, namely, that the CRISPR system shut down by AcrIB8 can be "rebooted" by controlling ARH expression. It has the following characteristics:

[0046] (1) High specificity: AcrIB8 only modifies Cas7b, does not induce broad-spectrum modification of host proteins, and has no cytotoxicity;

[0047] (2) Completely reversible: ARH can efficiently remove the modification and restore 100% of CRISPR activity;

[0048] (3) Catalytic regulation: Both AcrIB8 and ARH are enzymes, and high-intensity regulation can be achieved with low doses, with efficiency far exceeding that of traditional stoichiometric Acr;

[0049] (4) Clinical safety: It can be used as an "emergency brake switch" for gene editing, significantly improving the safety of gene drugs;

[0050] (5) Compatible with IB type system: long fragment deletion, low off-target effect, large capacity delivery, suitable for clinical gene therapy.

[0051] II. The sequences involved in the following embodiments:

[0052] 1. AcrIB8 (SEQ ID NO.1):

[0053] MKTIDRDEIAKDINACIKGLGRDIRTNWELGFEEGQIITLERYESWTTGGDFTVCNDCPVEYHFEIENEVPCHVVDYNNKEEVIALGAEDCEDENEVLLPAGTKLEVVYGEHEDDNEEMGFYTVIFKYLEEEK

[0054] 2. The IB-type CRISPR-Cas system consists of two elements: the Cascade complex and the Cas3b protein, wherein:

[0055] (1) The Cascade complex is composed of five protein subunits, Cas5b, 6b, 7b, 8b, 11b, and crRNA, combined in a ratio of 1:1:7:1:3:3. The sequences are as follows

[0056] Cas5b (SEQ ID NO.2):

[0057] MKLKERLMLIMKAIRVKLWQDLVNYKKPTSFQLKETYPLPPYSTVIGMIHTLCGFTSYHEMKISIQGKYFSKVNDLATRYEFKNGMTYDATRHQIKVDNYGVSRGISTVELLVDLELLLHIIPEDPALVPVIEKAFKEPIEYPSLGRREDIATIQAVDVVEVEKRKPKENKSVNISKDYNAYVPISLAESKVVRFKSQESSVGRSKLLGTRYLLTEKYERVNHGTEKAPKFFRKWRKKDVIYSSRIFVSKNDVFFLDKDDCLVFIEEEV

[0058] Cas6b (SEQ ID NO.3):

[0059] MGVTKMRLKINCDFESNIIPKDYRGKIVSLFKIGIEKSSPERYGDLFGSNKRKNYTFSIYLPKPKNNKNEIYLEEKNCIINFSTGDAETGIVFYNAFMYLRNKKVSFSSQNNITIKNIDIIREKKIISNKVYLKTLSPVVSRDHNKVTYKNWFYNFEDEEFEPTLKRNMLPFLIEEFGEQARFDVEKIKITPISMKKVVVYCHEIHIESSVGIFELTAEPYLQEYFLQNGLGSMTGSGFGMLEQL

[0060] Cas7b (SEQ ID NO.4):

[0061] MEETQMKNKGLAMTIVFQAESANYGESLGNISALKISRNNGDQYTYISRQAIRYNLMEQIGEKVAPVKAEGGGDKKVIQFLSEASIADFPELDFFGYLKTEKGTAGQKRSAKVRLSNAILETFKGDLDFLTNKGQADKINENMNIAQAEIHKSYYRYTITIDLDQIGIDGTVEIDNKEKARRVKKLMDTVAFLYRDIRGRREDLKPLFVIGGVYDVKNPVFQNVVDVLDNKIVIKNIDDLLQYEDIRENTKVGIIDGQFANADEVKSKLNAESVPAFFNEIKGKIDAYYEGN

[0062] Cas8b (SEQ ID NO.5):

[0063] MQAEIEVRANDWLINSGITGFLNIVGKENVRIDGQSLYFSTIDEGFETKYFNYFIKEYKETLAWHKIVSYKEKMEYYYRAEEFASFDEKALDDLNKYTKDVVKFYLKKPNYIKVFPLIDPEANITEWLGNLTTITISKKQKFEEVKVEILESVKSTYNQIDAIIDFCASEKGLKYLGAKNLIYSVINKGWSGVSFLFKQTKFIDPYEDYKTTFLDPVFEYLDTDLSKAKYNCFICNQPIKTLKLDLSFMNDVGFDTARKTSHVWDFNNDVATCPVCRLIYS CVPAGFTYVYGEGMFVNDSFSIDKLLDVNVHMRESILHFNNEGINSNNPYRALVESITMEKEDKRRYELADIQLVRYENEHYRFNILSKKMLHILNDSKTILKSLIRCGYKEGNNLNINLYKEVIQHLMNNENLFTLIHKL IFYKQTNANGLYYNMGHVAGILEINTKFLKEIDVMTNISQKQLWFVQSCGTEFKEGYYKKSENKIAGITYKLLNALKVNDKDGFMDTLLNSYSYLAKPIPSVFMNVFSNDEAFKSVGYAFMLGVGGERTKKEDGGNTDEK

[0064] Cas11b (SEQ ID NO.6):

[0065] MGHVAGILEINTKFLKEIDVMTNISQKQLWFVQSCGTEFKEGYYGKKSENKIAGITYKLLNALKVNDKDGFMDTLLNSYSYLAKPIPSVFMNVFSNDEAFKSVGYAFMLGVGGERTKKEDGGNTDEK

[0066] crRNA:

[0067] AUUUACAUUU CACAAUAAGU AAUUAAAACA AAAAUAAUUA GCUUGAACAG CCGUUUCAGUAUUCAUUUAC

[0068] (2)The Cas3b sequence is as follows:

[0069] Cas3b (SEQ ID NO.7):

[0070] MQKYLAKSNPPETIQEHTDNLLKNYQTLKKLYPEINIDWYLLELVCLLHDLGKMNRLFQKKLGNGSGVGKEIPHGYLSVAFVPYSKLEDLGYSEDEIQVVYQAIARHHERKKDFTEQEWETEIEKLSEQWETFFYERLADNADYSSEEIDEIYFYPEARIFEGEDTVEEIETFKNYVQLKGLLNRIDFAASAGIDVELENDFLQESMEHQLANFREKNAAADWNLLQKYMLQHQNENVVVIAETGMGKTEAGLLWLGNHKGFFTLPLRTAINAIYTRVTREIVTDKQAQRVGLLHSETYSQYLFHEENTEMEIDEYYTRTRQMSLPVTICTLDQLFDFVFRYAGFEHKLATLSYSKVIIDEIQMYSPDLL AYLILGLSYIDKFGGKFCVMTATLPGIVLDLLHDNGVDFIQPEEKFVSERVRHSVEVVHTEIDSAFIEPFAGNRILVICNTISKAKKIYSELKAMFPNKKVRLIHSQFIKKDRSKEEEIFEDGQKDNPESCIWVATQVVEASLDIDFDLLFTELSDINGLFQRMGRCYRNRALEVDTNVYVFDGGEKVCSGVGQFIDKTIFMNSKKALEKCNGALTEMKKMEIVEQIYSTEALKESEFYDELTQALNYVKSFDSYELDKKEVRSKFRNINSITAIPSTVWQENEEEITSYMEILAKSSKEVSKKEKMIARTNLAEFMLNIPDYLYKKSEGEVHRINRYETVIEFKCDYSEDIGIIMQEKQKESLFF

[0071] 3、ARH protein sequence

[0072] LsARH (SEQ ID NO.8):

[0073] MRGQEEIDKKQYQTLFLKERLFPCVLGAVIGDSLGVPVEFKSREYLKEKPVIDMIGNGTYKLPKGTWSDDSSLTFTLMESLIKGYDINSIANNMVCFLDNGYWTPFGEVFDIGNATREALLRYKRKRSAFGCGGAEIFDNGNGAIMRIMPLVFYYGKDFSFKTKNRITEEVTSITHAHPRSILGSHIYIELLQNLFANMDKDLAYKEMKKYILENYNDYPFGNELTYYTRILDEDLSDLNEEEIKSSGYVVDTLESAIWSFLTTDSYKEAVLKAVNLGGDTDTISFIAGSLAGIYYKLEQIPMEWIEQIIKKEEILNLQRFLDSLVKE

[0074] LmARH (SEQ ID NO.9):

[0075] MRGQEEIDKEQYQVLFIKERLIPCVLGAVIGDCLGVPVEFKDREYLKQNPIVEMIGYGTYNQPKGTWSDDSSLTFALMESLISGYDINRIVNNMVSFMDDGFWTPYGEVFDIGSVTRESLNRYKNGVSVFECGGKDNFDNGNGAIMRIMPLVFYLGKDFSFGKKNKITEEVTRITHAHPRSILGSYVYIELLQNLFANMDKKLAYEEMQNYIRKNYSDYPFKDELQYYNNILEGNLYELKESNIKSSGYVVDTLEASIWAFLTTNSYKEAVLKAVNLGGDTDTIAFITGSLAGIYYKMEQIPVNWIDQIAKKEDILNLCNRFIESLIKQ

[0076] SpARH (SEQ ID NO.10):

[0077] MIDLREDTWTLQLYAQRYKGLSPKNSRELQLRMEYDPLKPNLPTSGEEQNSKPEWLNTPPCLIPESESLDKAKGALVGLAIGDAIGTTLEFLPRDKLHVNDMVGGGPFRLQPGEWTDDTSMALCLAESYISAGRLDITLFREKLVRWYRHGENSSNGRCFDIGNTTRNALEQYLKHGASWFGN TEPETAGNAAIIRQAPTSIFRRKSLQRTFADSDSQSMATHCAPESMASCQFLGFILNYLINGSSREKAFSPHVMPLPVRVLLINAGEYKEKKRDEIRSSGYVIDTLEAAMWAVWNTDNFHDAILLAANLGDDADSVAATTGQIAGALYGYSNIPKPWLDKLVQQERISNLAEQLFYMAPEEDF

[0078] 4. The DNA double strands used in the following examples were formed by annealing the following single-stranded primers synthesized by bioengineering:

[0079] Target DNA CCG FAM (54 bp; 5'-FAM or 3'-FAM fluorescent label)

[0080] GTAAATCCGGAATACTGAAACGGCTGTTCAAGCTAATTATTTTTGTTTTAATTAG

[0081] Target DNA CCG R (54 bp; 5'-FAM or 3'-FAM fluorescent label)

[0082] GTAATTAAAACAAAAATAATTAGCTTGAACAGCCGTTTCAGTATTCCGGATTTA

[0083] Example 1: Discovery and verification of AcrIB8 possessing ADP-ribosyltransferase activity

[0084] (1) To screen for ARH sequences that may be compatible with AcrIB8 (ART), this embodiment performed homology alignment between AcrIB8 and the C-terminal domain of Tre1. Specifically, AlphaFold2 was used to predict the protein structure of AcrIB8, and structural alignment analysis was performed on multiple homologous sequences of AcrIB8 and the C-terminal domain of Tre1. The results showed that five independent homologous sequences of AcrIB8 (AcrIB8_1 to AcrIB8_5) and the C-terminal domain of Tre1 (Tre1 CTD The structural overlap of AcrIB8 sequences was examined. The alignment results showed that all AcrIB8 homologous sequences shared an overall folding pattern with Tre1. CTD Highly similar, both employing the typical α / β domain arrangement of the ART family. The secondary structure elements in the catalytic core region exhibit high conservation, especially those involved in NAD. + The active site residues for binding and catalytic reactions are identical between AcrIB8 and Tre1.

[0085] This experiment revealed the structural basis of AcrIB8 as an ADP-ribosyltransferase and its evolutionary association with the classic toxin Tre1. Using the known Tre1 as a structural template, comparison revealed that these three ARHs share high homology with Tre1 in terms of structural domains and conform to the regulatory pattern of the toxin-antitoxin system. Therefore, they were selected as candidate molecules for subsequent functional verification. The ARH proteins were selected from: LsARH (from Listeria seeligeri), LmARH (from Listeriamonocytogenes), and SpARH (from Serratia proteamaculans).

[0086] (2) We chose NAD + As a potential coenzyme substrate for AcrIB8, it was co-incubated with the Cascade complex or Cas3b protein in vitro, and then detected by Western blot using an ADPr-specific antibody. The modification signal was displayed by chemiluminescence.

[0087] The results showed that ( Figure 1 ), in NAD +In its presence, AcrIB8 can perform ADP-ribosylation modification on the Cascade complex but not the Cas3b protein, with the molecular weight of the modified band roughly corresponding to the Cas7b subunit of the Cascade complex. Coomassie gel mapping was used to determine the accuracy of the amount of each protein added in the reaction and the protein size corresponding to the modification site. These results demonstrate the substrate specificity (AcrIB8 modifies only the Cascade complex and not other irrelevant proteins) and target specificity of AcrIB8 (within the Cascade complex, AcrIB8 modifies only the Cas7b subunit and not other subunits such as Cas8b, Cas5b, Cas6b, and Cas11b).

[0088] Example 2

[0089] (1) ARH protein can reverse AcrIB8-mediated ART modification.

[0090] Example 1 has confirmed that AcrIB8, as an ADP-ribosyltransferase, can specifically modify the Cas7b subunit in the Cascade complex. To further investigate whether this modification process is reversible and whether ARH proteins from different species possess demodification activity, this invention reconstructed the demodification reaction system in vitro. The ADPr-Cascade complex was obtained by co-expressing Cascade and AcrIB8, and then incubated overnight on ice with three different ARH proteins (LsARH, LmARH, and SpARH). The reduction of modification signals was detected by Western blotting. The results of Western blotting showed ( Figure 2 In a reaction system using ADPr-Cascade as a substrate, the addition of LsARH, LmARH, and SpARH proteins resulted in varying degrees of attenuation of the ADP-ribosylation modification signal at the Cas7b position. The negative control group (without ARH) showed a stable modification signal, confirming that the signal reduction depended on the addition of ARH protein. Coomassie Brilliant Blue staining results showed consistent protein loading amounts across all lanes, ruling out the influence of loading amount differences on signal intensity.

[0091] To achieve co-expression of AcrIB8 and the Cascade complex in the same cell, we employed a multi-plasmid co-transformation strategy, as detailed below:

[0092] Carrier selection:

[0093] Cascade complex: Two vectors, pETDuet-1 and pACYCDuet-1, were used. pETDuet-1 carries ampicillin resistance, with its two multiple cloning sites (MCS1 and MCS2) containing the Cas8b-Cas5b genes (connected by the ribosome binding site (RBS) sequence of the T7 gene 10 to achieve bicistronic expression); pACYCDuet-1 carries chloramphenicol resistance, with its MCS1 and MCS2 containing the Cas7b-His6 and Cas6b genes, respectively.

[0094] AcrIB8: The AcrIB8 gene was cloned into MCS1 of the pRSFDuet-1 vector (carrying kanamycin resistance), and crRNA was inserted into MCS2.

[0095] Linkage sequences: All vectors utilize the T7 promoter, RBS, and T7 terminator elements inherent in the Duet series vectors. Genes are linked via RBS sequences, and each gene has independent transcription and translation initiation elements, thus ensuring independent and efficient expression of each protein.

[0096] Expression system: The above plasmids (pETDuet-1-Cas5b / 8b, pACYCDuet-1-Cas7b / 6b, pRSFDuet-1-crRNA (MCS2)-AcrIB8 (MCS1)) were co-transformed into E. coli BL21(DE3) competent cells.

[0097] Co-expression and purification: All proteins and crRNAs were simultaneously expressed and assembled in the host bacteria by IPTG induction. After lysis, the complex containing Cascade and possibly bound AcrIB8 was purified in one step by Ni-NTA affinity chromatography using the His tag on Cas7b.

[0098] In the co-expression system, we mainly observed the modifying effect of AcrIB8 on Cascade, but AcrIB8 and Cascade did not form a stable, co-purifiable complex (consistent with its enzymatic modification characteristics).

[0099] (2) ARH protein restores the DNA-binding activity of the Cascade complex after AcrIB8 modification.

[0100] Electrophoretic Mobility Shift Assay (EMSA), a classic technique widely used in molecular biology, is employed to detect interactions between proteins and nucleic acids (such as DNA or RNA). The principle is that after a protein specifically binds to DNA to form a complex, the increased molecular weight of the complex slows its migration rate in non-denaturing polyacrylamide gel electrophoresis, resulting in it lagging behind the free DNA band. By comparing the migration position with that of the free DNA band in the control group, it can be determined whether the target protein has specifically bound to a particular DNA sequence.

[0101] In this invention, the DNA probe is labeled with a FAM fluorescent group at its 5' end, facilitating subsequent band detection via fluorescence imaging. For example... Figure 3 As shown, in the negative control lane, free double-stranded DNA (dsDNA) was located at the bottom of the gel, presenting a single, clear band, providing a baseline reference for migration location in the experiment. In the positive control lane, after co-incubation of the unmodified natural Cascade complex with dsDNA, a significant hysteresis band appeared in the upper part of the gel, corresponding to the successfully formed Cascade-dsDNA complex; simultaneously, the signal of the free dsDNA band was significantly weakened, confirming that the Cascade complex used in this experiment possesses normal target DNA binding activity.

[0102] Three different ARH proteins were incubated with ADPr-modified Cascade complexes at a 1:10 molar ratio at 18°C ​​for 8 hours, followed by EMSA analysis. The results showed that the intensity of the free dsDNA band was significantly weaker than the control group, but the corresponding lag band signal of the Cascade-DNA complex was weak, indicating that the ARH proteins' recovery efficiency for Cascade complex DNA binding activity was poor under these conditions. Increasing the incubation temperature to 23°C and maintaining it for 8 hours revealed that the Cascade-DNA complex band intensity gradually increased in a time-dependent manner. This phenomenon confirms that ARH proteins can effectively restore the DNA binding activity of the Cascade complex inhibited by AcrIB8 through ADP-ribosylation modification.

[0103] Based on the combined experimental results, we can conclude that ARH proteins, by reversing AcrIB8-mediated ADP-ribosylation modification, relieve the functional inhibition of the Cascade complex, thereby restoring the core function of this complex in recognizing and binding target DNA. Furthermore, we conducted a parallel comparison of the effects of the three ARHs using electrophoretic migration assays (EMSA). The results showed that SpARH was the combination with the highest efficiency in restoring ADPr-Cascade DNA binding activity. Specifically, under the same molar ratio conditions, the SpARH group exhibited the brightest restored Cascade-DNA complex band, along with the greatest reduction in the free DNA probe band, indicating its superior demodification efficiency and functional restoration ability.

[0104] Example 3

[0105] (1) The presence of AcrIB8 can cause the inactivation of the IB-type CRISPR-Cas system in prokaryotes.

[0106] To verify the AcrIB8 inhibition mechanism and modification function, this embodiment conducted an in vivo enzyme digestion experiment at the prokaryotic level (Escherichia coli BL21(DE3) was selected in this embodiment). The results showed ( Figure 4 In the presence of AcrIB8, ADP-ribosylation modification of the Cascade complex significantly inhibited the cleavage activity of the type I-B CRISPR-Cas system (where Cas3b is cloned onto the pET28a plasmid) on target DNA (the target DNA sequence is cloned onto the pCDFDuet-1 plasmid; in the presence of the functional Cascade complex and Cas3 nuclease, they recognize and cleave this target DNA, leading to reporter plasmid loss and cell sensitization to the corresponding antibiotics (such as streptomycin / spectinomycin). The cleavage efficiency of the CRISPR system and the regulatory effect of AcrIB8 / ARH were quantified by comparing cell survival rates (colony-forming units) on different antibiotic plates. The inhibitory effect was concentration-dependent and the results were consistent across repeated experiments. In summary, this modification prevents the Cascade complex from binding to the DNA target, resulting in decreased affinity, conformational changes, and blocking the function of the Cas nuclease, thus preventing it from cleaving exogenous DNA.

[0107] (2) ARH achieves reversible regulation of CRISPR immune activity in prokaryotes.

[0108] The addition of ARH to a CRISPR-Cas system containing both AcrIB8 and type I-B cells showed a reversal effect (cleavage restoration). ARH protein reversed AcrIB8-mediated modification, restoring cleavage activity in the CRISPR system. Following induction, the number of colonies on the four-antibody plates decreased significantly again, as shown in the results. Figure 5 As shown in the figure, by comparing the differences in colony growth between groups with and without AcrIB8, the function of ARH proteins from different sources in reversing the inhibitory effect of AcrIB8 can be determined, thereby verifying their regulatory activity as "Anti-Anti-CRISPR" factors. When AcrIB8 is present, the CRISPR-Cas system is inactivated (unable to cleave exogenous DNA); however, after the introduction of ARH, the CRISPR system regains its ability to cleave target DNA, re-establishing immune defense. SpARH showed the most significant recovery and the most pronounced target DNA cleavage ability.

[0109] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A reversibly tunable complex for a CRISPR-Cas system, characterized in that, The reversibly modulated complex of the CRISPR-Cas system includes: An anti-CRISPR protein, the amino acid sequence of which is shown in SEQ ID NO.1; The reverse regulator of the anti-CRISPR protein, wherein the amino acid sequence of the reverse regulator of the anti-CRISPR protein is one of the sequences shown in SEQ ID NO. 8-10.

2. The nucleic acid sequence encoding the reversible regulatory complex of the CRISPR-Cas system as described in claim 1.

3. A recombinant plasmid carrying the nucleic acid sequence of claim 2.

4. A gene editing system, characterized in that, The gene editing system includes: Cascade complex, wherein the Cascade complex contains a Cas7b subunit; Cas protein; The CRISPR-Cas system reversibly modulated complex according to claim 1.

5. The gene editing system according to claim 4, characterized in that, The Cascade complex contains Cas5b subunit, Cas6b subunit, Cas7b subunit, Cas8b subunit, Cas11b subunit, and crRNA; And / or, the Cas protein includes the Cas3b protein.

6. The gene editing system according to claim 5, characterized in that, It must contain at least one of the following characteristics: (1) The amino acid sequences of the Cas5b subunit, Cas6b subunit, Cas7b subunit, Cas8b subunit and Cas11b subunit are shown in SEQ ID NO.2-6, respectively; (2) The amino acid sequence encoding the Cas protein is shown in SEQ ID NO.7; (3) The molar ratio of Cas5b subunit, Cas6b subunit, Cas7b subunit, Cas8b subunit, Cas11b subunit and crRNA is 1:1:(5-8):1:3:(2-4); (4) The molar ratio of Cascade complex to anti-CRISPR protein is 1:(0.01-10). (5) The molar ratio of the Cascade complex to the anti-CRISPR protein inverse regulator is 1:(0.01-10).

7. Recombinant cells containing the CRISPR-Cas system reversible regulatory complex of claim 1, the nucleic acid sequence of claim 2, the recombinant plasmid of claim 3, or the gene editing system of any one of claims 4-6.

8. The use of the CRISPR-Cas system reversible regulatory complex of claim 1, the nucleic acid sequence of claim 2, the recombinant plasmid of claim 3, the gene editing system of any one of claims 4-6, or the recombinant cell of claim 7 in the preparation of gene editing products or gene drugs.

9. The application according to claim 8, characterized in that, The gene editing product is used for reversible regulation of gene editing.

10. The use of any of the sequences shown in SEQ ID NO. 8-10 or sequences with a sequence similarity of more than 90% in the preparation of the reverse regulator of the anti-CRISPR protein shown in SEQ ID NO. 1.

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