Fusion proteins, compositions and their applications in improving the stability of repetitive sequence replication

Abstract

This invention relates to the field of biotechnology, specifically to fusion proteins, compositions, and their applications in improving the stability of repetitive sequence replication. The invention provides fusion proteins comprising anchoring proteins and stabilizing proteins that aid in chromatin silencing and / or DNA repair. It also provides compositions comprising fusion proteins and guide RNA designed for target repetitive sequences, and their applications in improving the stability of repetitive sequence replication. Specifically, the compositions, leveraging the epigenetic modification capabilities of Sir2 deacetylase and the binding ability of dCas9, regulate the higher-order structure of chromosomes through the expression of gRNA arrays and dCas9-Sir2 fusion proteins. They can bind to in vivo expressed gRNA arrays to target repetitive DNA sequences, helping to suppress sequence instability caused by recombination between repetitive DNA sequences, and providing a more comprehensive and less impactful stabilizing effect on chromosomes.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to fusion proteins, compositions, and their application in improving the stability of repetitive sequence replication. Background Technology

[0002] The synthesis and assembly of large DNA fragments is a key synthetic biology technology. With the continuous development of synthetic genome technology, the synthesis of Mb-level DNA has become possible, and the construction of artificially designed large-fragment chromosomes based on the genomes of microorganisms such as E. coli or Saccharomyces cerevisiae has become possible. In 2024, the research group of Professor Yuan Yingjin at Tianjin University used HAnDy technology to assemble a 1.024 Mb artificial chromosome sequence encoded by 542 exogenous genes in Saccharomyces cerevisiae. In 2023, the research group of Professor Jason W. Chin used Bacterial Artificial Chromosome Stepwise Insertion Synthesis (BASIS) to assemble a 1.1 Mb human chromosome.

[0003] The assembled sequences primarily use the genomes of various organisms as references, relying mainly on *E. coli* and *Saccharomyces cerevisiae* as tools for synthesis, assembly, and enrichment. Although assembling artificially designed chromosomes of higher organisms is now possible, large DNA fragments within *E. coli* and *Saccharomyces cerevisiae* are prone to rearrangement and deletion during their own growth and replication process. This is due to the inherent complexity of higher organism genomes and the large number of homologous repetitive sequences, which significantly limits the depth and length of fully artificially designed higher organism genomes. This is because important regions on chromosomes such as centromeres and telomeres, as well as proteins with important functions such as antibodies, are complex repetitive sequences, and there are currently no specific methods for stabilizing repetitive sequences. Existing methods for reducing homologous recombination capacity mainly focus on the gene level. Conventional methods for stabilizing repetitive sequences achieve this by knocking out or weakening homologous recombination genes, thereby reducing the activity of related recombination enzymes. However, this method also has adverse effects on the growth and development of the host organism itself. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a fusion protein, a composition and its application in improving the stability of repetitive sequence replication. The composition provided by the present invention utilizes the epigenetic modification ability of Sir2 deacetylase and the binding ability of dCas9 protein to regulate the higher-order structure of chromosomes through gRNA array, expression of dCas9 protein and Sir2 protein, thereby improving the stability of repetitive sequence replication by means of epigenetic modification.

[0005] This invention provides fusion proteins, including anchoring proteins and stabilizing proteins that aid in chromatin silencing and / or DNA repair, wherein:

[0006] The anchoring protein includes Cas proteins, and the Cas proteins include at least one of the dCas9 family proteins and the dCas12 family proteins;

[0007] The stable protein includes at least one of histone deacetylase, histone methyltransferase, and DNA methyltransferase.

[0008] Compared with existing technologies, the fusion protein provided by this invention, with the help of anchoring and stabilizing proteins, can better play its role in stabilizing repetitive DNA sequences, thereby achieving more accurate technical results.

[0009] In some embodiments, the anchoring protein is the dCas9 protein, and the amino acid sequence of the dCas9 protein is shown in SEQ ID NO:1;

[0010] The stable protein is yeast endogenous histone deacetylase 2, and the amino acid sequence of yeast endogenous histone deacetylase 2 is shown in SEQ ID NO:2.

[0011] The fusion protein provided by this invention, due to the presence of two silencing mutants of the RuvC1 and HNH nuclease domains (D10A and H841A) in the dCas9 system, forms a Cas9 with lost nuclease activity (dead Cas9, dCas9). Deletion of the Sir2 gene in *Saccharomyces cerevisiae* leads to increased acetylation of histones H3 and H4 in the yeast rDNA region, which plays a role in chromatin silencing and DNA repair. Therefore, this invention utilizes the non-cleaving, palindromic repeat sequence-associated protein system 9 as an anchoring protein, and leverages endogenous histone deacetylases in yeast to perform targeted epigenetic regulation of repetitive DNA sequences in *Saccharomyces cerevisiae*, thereby stabilizing repetitive DNA sequences and achieving more precise technical effects.

[0012] In some embodiments, fluorescent proteins and linking amino acids are also included.

[0013] In some specific embodiments, the fluorescent protein includes the gene for the BFP fluorescent reporter protein, which has the amino acid sequence shown in SEQ ID NO:3;

[0014] The linking amino acids include linking amino acid A and linking amino acid B. The anchoring protein and the fluorescent protein are linked through linking amino acid A, and the fluorescent protein and the stable protein are linked through linking amino acid B. Linking amino acid A has the amino acid sequence shown in SEQ ID NO:4, and linking amino acid B has the amino acid sequence shown in SEQ ID NO:5.

[0015] The fusion protein of the present invention can be composed of, from N-terminus to C-terminus, an anchoring protein, linking amino acid A, a fluorescent protein, linking amino acid B, and a stabilizing protein, or a stabilizing protein, linking amino acid B, a fluorescent protein, linking amino acid A, and an anchoring protein. The fusion proteins synthesized with the above linking order have equivalent effects.

[0016] In some specific embodiments, the fusion protein has an amino acid sequence as shown in SEQ ID NO:6.

[0017] The present invention provides a composition comprising the aforementioned fusion protein and a guide RNA designed for a target repetitive sequence.

[0018] In some embodiments, the guide RNA includes at least one of an sgRNA array, an siRNA array, and a miRNA array designed for the target repetitive sequence.

[0019] In some specific embodiments, the guide RNA is an sgRNA array, which includes at least one sgRNA transcription unit, wherein the sgRNA transcription unit, from the 5' end to the 3' end, is sequentially: promoter A-(tRNA-sgRNA-crRNA). n -tRNA-terminator A, n is an integer greater than or equal to 1, and the sequences of tRNA and sgRNA in the sgRNA transcription unit are different.

[0020] When dCas9 is co-expressed with sgRNA containing a 20-base-pair complementary region, a DNA recognition complex is generated. This complex binds only to the target DNA without inducing cleavage of the target gene. Compared with existing technologies, the composition provided by this invention allows dCas9 to recognize and bind to a specific 20bp nucleic acid sequence via its own gRNA. Therefore, it can uniformly target a wide range of repetitive sequence regions by transcribing a limited number of gRNAs. This invention constructs a gRNA array targeting the recognition sequence of the anchoring protein dCas9 and stabilizes repetitive DNA sequences in Saccharomyces cerevisiae by fusing expression of dCas9-SIR2 protein and the gRNA array.

[0021] In some embodiments, promoter A includes at least one of the pol II and pol III series promoters.

[0022] In some embodiments, terminator A is an AT-rich nucleotide sequence, and terminator A is 39 bp in length.

[0023] In some embodiments, n is 3 or 4.

[0024] In some specific embodiments, the target repetitive sequence is a synthetic immunoglobulin gene locus, and the sgRNA array includes 13 sgRNAs, the nucleotide sequences of which are shown in SEQ ID NO:7~19.

[0025] The present invention provides an expression module comprising a nucleic acid encoding the fusion protein and the guide RNA in the composition.

[0026] In the expression module provided by this invention, the nucleic acid encoding the fusion protein and the guide RNA can be expressed separately or ligated in the same vector and expressed together, with comparable effects; when ligated in the same vector, the ligation order of the nucleic acid encoding the fusion protein and the guide RNA has a comparable effect on stabilizing repetitive sequence DNA.

[0027] In some embodiments, at least one of promoter B, enhancer and terminator B is also included.

[0028] In some specific embodiments, it includes, in sequence: promoter B, enhancer, nucleic acid encoding the fusion protein, terminator B, and guide RNA.

[0029] Preferably, the promoter B comprises the natural promoter TDH3P, which has a nucleotide sequence as shown in SEQ ID NO:20;

[0030] The enhancer has a nucleotide sequence as shown in SEQ ID NO:21;

[0031] The terminator B has a nucleotide sequence as shown in SEQ ID NO:22.

[0032] The present invention provides a recombinant vector, including the expression module and the backbone vector.

[0033] The present invention provides host cells for transformation or transfection of the recombinant vector.

[0034] This invention provides at least one of the following (1) to (4) applications in improving the replication stability of repetitive sequences:

[0035] (1) The fusion protein;

[0036] (2) The composition described above;

[0037] (3) The aforementioned expression module;

[0038] (4) The recombinant vector;

[0039] (5) The host cell.

[0040] This invention provides a drug and / or kit for improving the replication stability of repetitive sequences, comprising at least one of the following ① to ④:

[0041] ① The fusion protein

[0042] ② The aforementioned composition;

[0043] ③ The aforementioned expression module;

[0044] ④ The aforementioned recombinant vector;

[0045] ⑤ The host cell mentioned above.

[0046] The present invention provides a method for improving the replication stability of repetitive sequences, comprising adding the drug and / or kit to a test sample.

[0047] This invention provides a fusion protein comprising an anchoring protein and a stabilizing protein that aids in chromatin silencing and / or DNA repair. It also provides a composition comprising the fusion protein and guide RNA designed for target repetitive sequences, and its application in improving the replication stability of repetitive sequences. The composition, leveraging the epigenetic modification capabilities of Sir2 deacetylase and the binding ability of dCas9, regulates the higher-order structure of chromosomes through the expression of gRNA arrays and the dCas9-Sir2 fusion protein. It can bind to the in vivo expressed gRNA arrays to target repetitive DNA sequences, promoting histone deacetylation of the target repetitive DNA sequences to form dense heterochromatin regions. This helps suppress sequence instability caused by recombination between repetitive DNAs, providing a more comprehensive and less impactful stabilizing effect on the chromosome itself. Utilizing an epigenetic modification system for coupling provides a novel approach to targeted binding for the stabilization of repetitive sequences. Based on the composition provided by this invention, a stable plasmid was designed for a 120Kb repetitive sequence, capable of stable replication for over 30 generations. Attached Figure Description

[0048] Figure 1 This is a schematic diagram showing the composition of each unit in the repeating sequence in Example 1;

[0049] Figure 2 A schematic diagram showing gRNA targeting a wide range of repetitive sequence regions;

[0050] Figure 3 This shows the specific shape of the gRNA array;

[0051] Figure 4 The recombinant plasmid map in Example 2 is shown;

[0052] Figure 5 This shows the positive result of PCR verification of the 120Kb repeat sequence interface primers in Example 3;

[0053] Figure 6 This shows the full PCR interface verification results of the fifth-generation strain containing a stable plasmid in Example 3;

[0054] Figure 7 The PCR interface verification results of the fifth-generation strain containing the stable plasmid in Example 3 after 7 generations in a medium without hygromycin are shown.

[0055] Figure 8 The PCR interface verification results of the fifth-generation strain containing the stable plasmid in Example 3 after 7 generations in a medium containing hygromycin are shown.

[0056] Figure 9 The results of full PCR interface verification of the 30th generation strain containing a stable plasmid in Example 3 are shown. Detailed Implementation

[0057] This invention provides fusion proteins, compositions, and their applications in improving the stability of repetitive sequence replication. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0058] The purpose of this invention is to utilize the deadclustered regularly interspaced short palindromic repeats associated proteins (dCas9) as an anchoring module, and to use histones and yeast endogenous histone deacetylase 2 (SIR2) to perform targeted epigenetic regulation of repetitive DNA sequences in Saccharomyces cerevisiae.

[0059] The CRISPR / Cas9 system consists of the Cas9 protein and gRNA. The Cas9 protein contains two main nuclease domains—the RuvC domain and the HNH domain. The former cleaves non-complementary DNA strands, and the latter cleaves complementary DNA strands. The gRNA is a chimeric RNA formed by the binding of trans-activated cripr RNA (tracrRNA) to specific cripr ribonucleic acid (crRNA), which guides Cas9 to its target. Current research fuses crRNA and tracrRNA into a single RNA, called sgRNA (single-guide RNA). This modified CRISPR / Cas9 system has become the preferred tool for gene editing. The dCas9 system contains two silent mutants, the RuvC1 and HNH nuclease domains (D10A and H841A), forming a Cas9 with lost nuclease activity (dead Cas9, dCas9). When dCas9 is co-expressed with sgRNA containing a 20-base-pair complementary region, a DNA recognition complex is generated. This complex binds only to the target DNA without inducing cleavage of the target gene.

[0060] Sir2 in *Saccharomyces cerevisiae* is the earliest discovered member of the Sir2 protein family. This protein contains a conserved catalytic region of approximately 275 amino acids, and histone deacetylation is the main biological function of Sir2 in *Saccharomyces cerevisiae*. Deletion of the Sir2 gene leads to increased acetylation of histones H3 and H4 in the yeast rDNA region. This plays a role in chromatin silencing and DNA repair.

[0061] Because dCas9 can recognize and bind to specific 20bp nucleic acid sequences using its own gRNA, it can uniformly target a large range of repetitive sequence regions by transcribing a limited number of gRNAs. This technology constructs a gRNA array targeting the dCas9 protein's recognition sequence and then uses the fusion expression of the dCas9-SIR2 protein and the gRNA array to stabilize repetitive DNA sequences in *Saccharomyces cerevisiae*.

[0062] The compositions and methods for improving the replication stability of repetitive sequences provided by this invention have broad applicability, and gRNA arrays can be designed according to different repetitive fragments to achieve stable inheritance of repetitive sequences. However, due to the large amount of time and resources required in practice, this invention cannot show them all.

[0063] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0064] Example 1: gRNA array design targeting specific repetitive sequences

[0065] The specific repetitive sequence constructed in this embodiment is a synthetic immunoglobulin locus (its sequence information can be found in the National Biotec Group bioproject project number PRJCA030768, accessible at https: / / ngdc.cncb.ac.cn / bioproject / browse / PRJCA030768, where the resource name CRA019373 in the project's data information is the nucleotide sequence of the synthetic immunoglobulin locus in this application). This locus was designed using a modular approach. This design feature reduces intergenic regions, but at the cost of highly repetitive sequences, with extremely high similarity between modules, forming an intermittent repetitive structure similar to the (AB)60 pattern. A schematic diagram of each unit of the repetitive sequence is shown below. Figure 1 As shown in the figure. Clustal Omega analysis revealed that the minimum pairwise similarity among the 60 V Regions was 51.55%, the average similarity was 71.47%, and the highest was 99.67%. In our study, an allele clustering scheme was used. Multiple V gene Regions, each approximately 300 bp long and differing only in single nucleotide polymorphisms (SNPs), were separated by spacers of approximately 1.5 kb.

[0066] For this complex sequence, we designed it using software (https: / / github.com / dudushou / SynIG). Its principle is to generate gRNAs targeting repetitive sequence regions, aiming to ensure that a limited number of gRNAs can uniformly target a large area of ​​repetitive sequence regions. A schematic diagram illustrating gRNA targeting a large area of ​​repetitive sequence regions is shown below. Figure 2 As shown in Table 1, gRNAs meeting the requirements were screened by searching for kmers containing 20 bp+ NGG in repetitive sequences and sorting the number of kmers containing NGG. Finally, the screened gRNAs were combined to ensure that the gap between any two gRNA targets did not exceed 2 kbp. The sgRNA sequences are shown in Table 1 below.

[0067] Table 1

[0068]

[0069] The specific shape of the gRNA array is as follows: Figure 3As shown, the gRNA array includes multiple transcription units, each containing a pol promoter, one or more tRNAs, three sgRNAs, a crRNA, a tRNA, and a terminator array. The pol promoter in the gRNA array includes pol II and pol III series promoters, with pol II promoters including pol II-1-9. The tRNAs in the gRNA array contain tRNAs used by 61 codons.

[0070] Example 2 Plasmid Construction

[0071] A dCas9-Sir2 fusion protein was designed for epigenetic modification to stabilize long repetitive DNA sequences. The fusion protein comprises: dCas9 protein, BFP fluorescent reporter protein, and Sir2, an endogenous deacetylated protein from Saccharomyces cerevisiae. The specific sequences are shown below:

[0072]

[0073] The amino acid sequence of Sir2 in the fusion protein is (SEQ ID NO: 2): MTIPHMKYAVSKTSENKVSNTVSPTQDKDAIRKQPDDIINNDEPSHKKIKVAQPDSLRETNTTDPLGHTKAALGEVASMELKPTNDMDPLAVSAASVVSMSNDVLKPETPKGPIIISKNPSNGIFYGPSFTKRESLNARMFLKYYGAHKFLDTYLPEDLNSLYIYYLIKLLGFEVKDQALIGTINSIVHINSQERVQDLGSAISVTNVEDPLAKKQTVRLIKDLQRAINKVLCTRLRLSNFFTIDHFIQKLHTARKILVLTGAGVSTSLGIPDFRSSEGFYSKIKHLGLDDPQDVFNYNIFMHDPSVFYNIANMVLPPEKIYSPLHSFIKMLQMKGKLLRNYTQNIDNLESYAGISTDKLVQCHGSFATATCVTCHWNLPGERIFNKIRNLELPLCPYCYKKRREYFPEGYNNKVGVAASQGSMSERPPYILNSYGVLKPDITFFGEALPNKFHKSIREDILECDLLICIGTSLKVAPVSEIVNMVPSHVPQVLINRDPVKHAEFDLSLLGYCDDIAAMVAQKCGWTIPHKKWNDLKNKNFKCQEKDKGVYVVTSDEHPKTL*;

[0074] The amino acid sequence of the BFPTAG tag in the fusion protein is (SEQ ID NO: 3): SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDLPSKLGHKLN;

[0075] The amino acid sequence of the fusion protein is (SEQ ID NO: 6):

[0076]

[0077] The fusion protein and gRNA array were constructed on a single plasmid to function. The expression of the dCas9-Sir2 fusion protein was first driven by the endogenous strong promoter (TDH3P) in Saccharomyces cerevisiae, and the transcription was terminated by the synthetic terminator Tsynth8 after binding to the 5' enhancer. Subsequently, the gRNA array sequence was loaded and the two sequences were integrated into the pRS42H plasmid. After the synthesis and verification of the correctness of the treatment, it was transformed into Saccharomyces cerevisiae strains by acetic acid transformation, and it was able to remain stable in the medium containing hygromycin.

[0078] The DNA sequence of the natural promoter TDH3P is (SEQ ID NO: 20): ;

[0079] The 5' end enhancer sequence is (SEQ ID NO: 21): AaaaaaAGGGAGCCCAAAA;

[0080] The sequence of the synthesis terminator Tsynth8 is (SEQ ID NO: 22): TATATAAACTCATTTACTTATGTAGGAATAAAGAGTATCATCTTTCAAA;

[0081] The linking amino acids between the dCas9 protein and TagBFP are (SEQ ID NO: 4): AYPYDVPDYASLGSGSPKKKRKVEDPKKKRKVDGIGSGSNGSSGS. Positions 2 to 10 are the human influenza hemagglutinin (HA) tag, and positions 17 to 23 and 26 to 32 are two identical SV40 NLS nuclear localization sequences.

[0082] The linking amino acid between TagBFP and Sir2 protein is (SEQ ID NO: 5): GGGGGMDAKSLTAWS.

[0083] Specific plasmid maps are shown below. Figure 4 As shown.

[0084] Example 3 Stability Verification

[0085] We transformed the plasmid composed of the aforementioned 120kb repetitive fragment into a strain carrying pRS42H-dCas9-Sir2 using lithium acetate transformation. Simultaneously, as a control group, we transformed the same DNA fragment into the BY4742 yeast strain, which does not contain a stable plasmid. Furthermore, we designed 30 pairs of interface primers to cover the entire repetitive sequence and ensure its uniformity for the repetitive fragment. Subsequent validation experiments verified the replication stability of the repetitive fragment by amplifying the corresponding interfaces. Specific interfaces and their lengths are shown in Table 2 below. The interface validation results are as follows: Figure 5 As shown.

[0086] Table 2

[0087]

[0088] No strains with fully correct PCR interfaces were obtained from *Saccharomyces cerevisiae* strains lacking stable plasmids, while strains with fully correct interfaces appeared from *Saccharomyces cerevisiae* strains containing stable plasmids. These strains were passaged using a streak-transfer method, with each generation lasting 24 hours. Hygromycin was no longer added during the transfer process in the 5th generation, making it easier for the stable plasmid to be lost during culture. After one week (7 generations), the PCR interfaces of strains that lost the stable plasmid disappeared, while strains still cultured in hygromycin (i.e., possessing the stable plasmid) were still able to stably carry plasmids containing 120 kb repeat sequences. In our tests, strains containing stable plasmids were still able to carry 120 kb repeat sequences even after 30 generations.

[0089] The fifth-generation strain containing a stable plasmid has been verified to have a correct full PCR interface, such as... Figure 6 As shown.

[0090] After culturing the fifth-generation strain, it was cultured in a medium without hygromycin to cause the loss of the stable plasmid. After 7 generations, most of the PCR interface had been lost. Interface verification results are as follows. Figure 7 As shown.

[0091] Strains cultured in hygromycin-containing medium after the fifth generation did not lose stable plasmids. After seven generations, PCR results remained stable, similar to the fifth generation. Interface verification results were as follows... Figure 8 As shown.

[0092] Figure 9 The image in the middle is a PCR gel image of a strain containing a stable plasmid after 30 generations of culture. It shows that Saccharomyces cerevisiae can carry a 120 kb repeat sequence plasmid for more than 30 generations when it contains a stable plasmid, and the repeat sequence is relatively stable in Saccharomyces cerevisiae.

[0093] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that, This includes anchoring proteins and stabilizing proteins that help with chromatin silencing and / or DNA repair, among which: The anchoring protein includes the dCas9 protein in the Cas protein, and the amino acid sequence of the dCas9 protein is shown in SEQ ID NO:1; The stable protein includes yeast endogenous histone deacetylase 2, a protein deacetylase, and the amino acid sequence of yeast endogenous histone deacetylase 2 is shown in SEQ ID NO:

2.

2. The fusion protein according to claim 1, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, a sequentially linked anchoring protein, linking amino acid A, a fluorescent protein, linking amino acid B, and a stabilizing protein, wherein: The fluorescent protein has the amino acid sequence shown in SEQ ID NO:3, the linking amino acid A has the amino acid sequence shown in SEQ ID NO:4, the linking amino acid B has the amino acid sequence shown in SEQ ID NO:5, and the fusion protein has the amino acid sequence shown in SEQ ID NO:

6.

3. The composition, characterized in that, Includes the fusion protein of claim 1 or 2 and a guide RNA designed for the target repetitive sequence, wherein: The guide RNA comprises an sgRNA array, which includes at least one sgRNA transcription unit, wherein the sgRNA transcription unit, from the 5' end to the 3' end, is sequentially: promoter A-(tRNA-sgRNA-crRNA). n -tRNA-terminator A, n is an integer greater than or equal to 1, and the sequences of tRNA and sgRNA in the sgRNA transcription unit are different; Preferably, promoter A includes at least one of the pol II and pol III series promoters, terminator A is an AT-rich nucleotide sequence, and terminator A has a length of 39 bp.

4. The composition according to claim 3, characterized in that, The target repetitive sequence is a synthetic immunoglobulin gene locus, and the sgRNA array includes 13 sgRNAs, the nucleotide sequences of which are shown in SEQ ID NO:7~19.

5. An expression module, characterized in that, Includes the nucleic acid encoding the fusion protein of claim 1 or 2 and the guide RNA in the composition of claim 3 or 4.

6. A recombinant vector, characterized in that, It includes the expression module and skeleton carrier as described in claim 5.

7. Transform or transfect the host cell of the recombinant vector of claim 6.

8. At least one of the following (1) to (4) is used to improve the replication stability of repetitive sequences: (1) The fusion protein according to claim 1 or 2; (2) The composition according to claim 3 or 4; (3) The expression module as described in claim 5; (4) The recombinant vector according to claim 6; (5) The host cell as described in claim 7.

9. A drug and / or kit for improving the replication stability of repetitive sequences, characterized in that, Includes at least one of the following ① to ④: ① The fusion protein according to claim 1 or 2 ② The composition according to claim 3 or 4; ③ The expression module as described in claim 5; ④ The recombinant vector as described in claim 6; ⑤ The host cell as described in claim 7.

10. A method for improving the replication stability of repetitive sequences, characterized in that, Add the drug and / or kit as described in claim 9 to the test sample.

Images