Genome editing and detecting system for recognizing DNA target based on TDP-43 dimerization and application of genome editing and detecting system

Abstract

The invention discloses a genome editing and detecting system for recognizing a DNA target based on TDP-43 dimerization and application of the genome editing and detecting system, and belongs to the technical field of molecular biology. FokT fusion protein construction, cell transfection and functional labeling, FokT-Seq detection through high-throughput sequencing, target annotation and functional analysis and disease mutation experience verification are carried out, and the genome editing and detecting system for recognizing the DNA target based on TDP-43 dimerization is obtained. Specific recognition and functional marking on the TDP-43 dimerization dependent DNA binding site are achieved for the first time, and traditional ChIP-Seq, CUTamp, CCTP-Seq, CCTP-Seq, CCTP-Seq, CCTP-Seq; a conformational state monomer or dimer when TDP-43 is combined with DNA cannot be judged by methods such as FOkI, FOkI is only activated in a dimerization state, and only a DNA target of TDP-43 combined in a dimer form is marked, so that the structure specificity of recognition is remarkably improved, and false positive binding site interference is avoided.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a genome editing and detection system based on TDP-43 dimerization to recognize DNA targets and its application. Background Art

[0002] Transactive DNA-binding protein 43 (TDP-43) is an important RNA / DNA binding protein that plays a key role in multiple intranuclear processes, including RNA pre-splicing, mRNA stability regulation, and transport. In recent years, numerous studies have found that TDP-43 dysfunction is an important pathogenic factor in multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and Alzheimer's disease (AD). Its abnormalities manifest as loss from the nucleus, aggregation in the cytoplasm, and decreased protein dimerization ability. Among them, the loss of TDP-43 dimerization ability is considered to be an early key event that induces its dysfunction and pathological aggregation.

[0003] In the prior art, the methods for studying TDP-43 DNA binding sites mainly include the following categories:

[0004] 1. Chromatin immunoprecipitation (ChIP-Seq) technology:

[0005] ChIP-Seq is currently the most commonly used technique for studying DNA-binding protein targets, including TDP-43. It uses antibodies to enrich protein-DNA complexes and then performs high-throughput sequencing to obtain binding site information. However, this method has the following drawbacks:

[0006] 1) Unable to distinguish the protein binding state (monomer, dimer or higher form);

[0007] 2) Highly antibody-dependent and susceptible to nonspecific binding interference;

[0008] 3) Low spatial resolution, unable to accurately locate the cutting point;

[0009] 4) Failure to provide direct evidence of functional binding.

[0010] 2. Derivative technologies such as CUT&RUN and CUT&TAG:

[0011] These technologies have improved the resolution and background noise of ChIP-Seq to a certain extent, and improved the detection accuracy by enzyme-binding DNA segments. However, the following problems still exist:

[0012] 1) Like ChIP-Seq, it is unable to distinguish between protein binding states;

[0013] 2) Enzyme digestion efficiency depends on multiple conditions and is difficult to standardize;

[0014] 3) It is not possible to verify whether binding leads to functional changes (such as transcriptional activation or repression);

[0015] 4) It does not have operational functional labels and cannot serve as the basis for subsequent functional experiments.

[0016] 3. FokI-based DNA cleavage systems

[0017] FokI is an artificial nuclease whose cleavage activity depends on dimerization. It is commonly used in dual-enzyme cleavage strategies in systems such as CRISPR and TALEN. FokI itself lacks DNA binding ability and is typically fused to specific DNA-binding domains, such as ZFNs and dCas9. However, there are currently no published reports on the fusion of FokI with TDP-43 to examine its DNA binding ability or biological function.

[0018] In addition, studies have shown that the N-terminal domain (NTD) of TDP-43 mediates its dimerization or oligomerization, which plays a decisive role in its RNA splicing function and subcellular localization. However, in existing research, there is no technology that can specifically identify TDP-43 binding events in the "dimer state" and DNA, which poses a serious limitation in analyzing its regulatory mechanisms and pathological changes. Summary of the Invention

[0019] Technical issues solved:

[0020] The present application addresses the deficiencies of the prior art, and solves the deficiencies in the prior art such as the inability to distinguish whether TDP-43 binds to DNA in the form of a dimer, the inability to specifically identify whether TDP-43 binds to DNA in the form of a dimer, and the inability to achieve high-throughput detection of its functional targets. Furthermore, there is a lack of specific tools that can be used to mark its functional DNA binding sites, which leads to significant difficulties in studying the relationship between its dimerization function and downstream transcriptional regulation or disease mechanisms. The present application provides a genome editing and detection system based on TDP-43 dimerization to recognize DNA targets and its application, based on FokI nuclease and TDP -43 fusion constructed dimerization-dependent genomic target recognition system and its application in TDP-43 function research and neurodegenerative disease mechanism exploration, by constructing a fusion protein of TDP-43 and FokI nuclease cleavage domain, FokI enzyme cleavage is activated only when TDP-43 forms a dimer and binds to DNA, making FokI enzyme cleavage activity dependent on TDP-43 dimerization, thereby achieving specific recognition and genome-wide localization of dimer-dependent binding sites, and combining high-throughput sequencing technology for positioning and annotation, solving the technical problem that existing methods cannot specifically detect the functional binding state of TDP-43.

[0021] Technical solution:

[0022] To achieve the above objectives, this application is implemented through the following technical solutions:

[0023] A genome editing and detection system based on TDP-43 dimerization to recognize DNA targets, the operating steps are as follows:

[0024] The first step is to construct a FokT fusion protein: the FokT fusion protein includes the full-length TDP-43 protein or its functional domain with dimerization ability; the full-length TDP-43 protein or its functional domain with dimerization ability is connected to the cleavage domain of the FokI nuclease via a flexible linker peptide to ensure spatial conformational freedom;

[0025] The second step is cell transfection and functional labeling: the encoding plasmid of the FokT fusion protein is transfected into the target cells, and the labeled double-stranded oligonucleotide dsODN is simultaneously introduced so that subsequent double-strand break events are specifically inserted into the tag;

[0026] The third step is high-throughput sequencing (FokT-Seq): genomic DNA from transfected cells is extracted, dsODN is enriched, and a library is constructed. Whole-genome sequencing is then performed using a high-throughput sequencing platform. Bioinformatics methods are used to align the inserts with the reference genome to precisely locate the genomic location of double-strand breaks, thereby identifying target DNA sites for TDP-43 dimer binding.

[0027] Step 4: Target annotation and functional analysis: Combining existing gene annotation information and RNA sequencing data, further evaluate the transcriptional regulatory function of TDP-43 dimer binding targets;

[0028] Step 5: Disease mutant validation: Use TDP-43 mutants to construct a control FokT system and verify whether the mutation affects TDP-43 dimerization and its binding function by comparing the number and distribution of double-strand break events.

[0029] Furthermore, the functional domain with dimerization ability is the N-terminal domain.

[0030] Furthermore, the cleavage domain of the FokI nuclease, catalytic domain, does not contain its own DNA recognition domain.

[0031] Furthermore, the first step of constructing the FokT fusion protein includes a tag sequence for immunodetection or purification, and the tag sequence is FLAG and / or HA.

[0032] Furthermore, after the first step FokT fusion protein is expressed in cells, only when TDP-43 binds to the target DNA site in a dimer state, the two FokI cleavage domains approach to form a dimer, thereby activating its enzymatic cleavage function and generating detectable double-stranded DNA breaks at the binding site.

[0033] Furthermore, the target cells in the second step are one or more of 293T, neurons, and iPSC-derived neural cells.

[0034] Furthermore, in the second step, the dsODN carries a known sequence tag and can be integrated into the genomic break at the FokI restriction site, thereby enabling subsequent high-throughput detection.

[0035] Furthermore, the fourth step can analyze whether it is located in the gene promoter or enhancer region, or whether it overlaps with splicing regulation or disease-related exons.

[0036] Furthermore, in the fifth step, the TDP-43 mutant is ΔNTD or the pathology-related mutation M337V.

[0037] Application of a genome editing and detection system based on TDP-43 dimerization to recognize DNA targets in the study of DNA functions of RNA-binding proteins.

[0038] Principle explanation: This application is based on the genome editing and detection system and its application of TDP-43 dimerization recognition DNA target, and constructs an expression system (FokT) that fuses TDP-43 and FokI cleavage domain to achieve TDP-43 dimerization-dependent enzymatic cleavage activity; utilizes the dimerization dependence of FokI to achieve specific recognition of TDP-43 dimer-bound DNA sites; combines dsODN with GUIDE-seq technology to achieve whole-genome high-throughput detection of FokT cleavage targets (FokT-Seq); can be used to identify functional differences in TDP-43 disease-related mutants and reveal its pathogenic mechanism in diseases such as ALS; and provides an expandable and general platform for the study of DNA function of RNA-binding proteins.

[0039] Beneficial effects:

[0040] This application provides a genome editing and detection system based on TDP-43 dimerization to recognize DNA targets and its application, which has the following beneficial effects compared with the existing technology:

[0041] 1. High specificity: The FokT system and its accompanying FokT-Seq method recognize only DNA sites where TDP-43 binds in dimer form. For the first time, the system achieves specific identification and functional labeling of TDP-43 dimerization-dependent DNA binding sites. Traditional methods such as ChIP-Seq and CUT&RUN cannot determine the conformational state (monomer or dimer) of TDP-43 when bound to DNA. This method, however, leverages the fact that FokI is only activated in the dimerized state and labels only DNA targets bound by TDP-43 in dimer form, significantly improving the structural specificity of identification and avoiding interference from false-positive binding sites.

[0042] 2. High throughput: Combined with the GUIDE-seq method, genome-wide target localization can be achieved;

[0043] 3. Clear function: The enzyme cleavage event itself has functional significance, which is different from the traditional method of only enriching binding sites;

[0044] 4. Can be combined with mutant models: suitable for evaluating the effects of different mutants on TDP-43 dimerization binding ability;

[0045] 5. Strong adaptability: can be widely applied to a variety of cell models, including neurons and human iPSC systems;

[0046] 6. Accurate detection of functional binding sites: FokT-mediated DNA cleavage can be captured by the dsODN tag and mapped by sequencing, enabling direct recording of TDP-43 functional binding events at the cellular level, significantly superior to the indirect inference of traditional enrichment methods;

[0047] 7. Promote TDP-43 pathological mechanism research and target development: Combined with RNA-seq and other data, the binding sites detected by this invention can be located in the promoters or splicing regions of downregulated genes caused by TDP-43 regulatory imbalance, providing technical support for the study of the pathogenic mechanisms and the screening of therapeutic targets for diseases such as ALS and FTLD;

[0048] 8. Simple operation and strong adaptability: This method is based on conventional molecular cloning, cell transfection and second-generation sequencing processes. The experimental conditions are mature, no expensive equipment is required, and it can be widely applied to different types of mammalian cells, with good reproducibility and scalability. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples are conventional methods generally known to those skilled in the art and do not constitute any limitation to the present invention in any way.

[0050] Example 1:

[0051] A genome editing and detection system based on TDP-43 dimerization to recognize DNA targets, the operating steps are as follows:

[0052] The first step is to construct a FokT fusion protein: the FokT fusion protein includes a full-length TDP-43 protein or a functional domain thereof with dimerization ability, wherein the functional domain with dimerization ability is the N-terminal domain, and the cleavage domain of the FokI nuclease, which does not contain its own DNA recognition domain; the full-length TDP-43 protein or the functional domain thereof with dimerization ability is connected to the cleavage domain of the FokI nuclease by a flexible connecting peptide to ensure spatial conformational freedom; the first step of constructing the FokT fusion protein includes a tag sequence for immunodetection or purification, wherein the tag sequence is FLAG and / or HA; after the FokT fusion protein is expressed in cells, only when TDP-43 binds to the target DNA site in a dimer state, the two FokI cleavage domains approach to form a dimer, thereby activating its enzymatic cleavage function and generating a detectable double-stranded DNA break at the binding site;

[0053] The second step is cell transfection and functional labeling: the encoding plasmid of the FokT fusion protein is transfected into target cells. In the second step, the target cells are one or more of 293T, neurons, and iPSC-derived neural cells. A labeled double-stranded oligonucleotide dsODN is simultaneously introduced so that subsequent double-strand break events can be specifically inserted into the tag. In the second step, the dsODN carries a known sequence tag and can be integrated into the genomic break at the FokI restriction site, thereby enabling subsequent high-throughput detection.

[0054] The third step is high-throughput sequencing (FokT-Seq): genomic DNA from transfected cells is extracted, dsODN is enriched, and a library is constructed. Whole-genome sequencing is then performed using a high-throughput sequencing platform. Bioinformatics methods are used to align the inserts with the reference genome to precisely locate the genomic location of double-strand breaks, thereby identifying target DNA sites for TDP-43 dimer binding.

[0055] Step 4: Target Annotation and Functional Analysis: Combining existing gene annotation information and RNA sequencing data, further evaluate the transcriptional regulatory function of TDP-43 dimer-binding targets. This step can analyze whether they are located in gene promoters or enhancer regions, or whether they overlap with splicing regulation or disease-related exons.

[0056] Step 5: Disease mutant validation: A control FokT system is constructed using TDP-43 mutants. The TDP-43 mutants in the fifth step are ΔNTD or the pathology-associated mutation M337V. By comparing the number and distribution of double-strand break events, it is verified whether the mutation affects TDP-43 dimerization and its binding function.

[0057] The above is an exemplary description of the present invention. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by other skilled in the art without expending creative labor falls within the scope of protection of the present invention.

Claims

1. A genome editing and detection system based on TDP-43 dimerization to recognize DNA targets, characterized in that: The steps are as follows: The first step is to construct a FokT fusion protein: the FokT fusion protein includes the full-length TDP-43 protein or its functional domain with dimerization ability; the full-length TDP-43 protein or its functional domain with dimerization ability is connected to the cleavage domain of the FokI nuclease via a flexible linker peptide to ensure spatial conformational freedom; The second step is cell transfection and functional labeling: the encoding plasmid of the FokT fusion protein is transfected into the target cells, and the labeled double-stranded oligonucleotide dsODN is simultaneously introduced so that subsequent double-strand break events are specifically inserted into the tag; The third step is high-throughput sequencing (FokT-Seq): genomic DNA from transfected cells is extracted, dsODN is enriched, and a library is constructed. Whole-genome sequencing is then performed using a high-throughput sequencing platform. Bioinformatics methods are used to align the inserts with the reference genome to precisely locate the genomic location of double-strand breaks, thereby identifying target DNA sites for TDP-43 dimer binding. Step 4: Target annotation and functional analysis: Combining existing gene annotation information and RNA sequencing data, further evaluate the transcriptional regulatory function of TDP-43 dimer binding targets; Step 5: Disease mutant validation: Use TDP-43 mutants to construct a control FokT system and verify whether the mutation affects TDP-43 dimerization and its binding function by comparing the number and distribution of double-strand break events.

2. The genome editing and detection system based on TDP-43 dimerization to recognize DNA targets according to claim 1, characterized in that: The functional domain with dimerization ability is the N-terminal domain.

3. The genome editing and detection system based on TDP-43 dimerization recognition of DNA targets according to claim 1, characterized in that The cleavage domain of the FokI nuclease does not contain its own DNA recognition domain.

4. The genome editing and detection system based on TDP-43 dimerization to recognize DNA targets according to claim 1, characterized in that The first step of constructing the FokT fusion protein includes a tag sequence for immune detection or purification, and the tag sequence is FLAG and / or HA.

5. The genome editing and detection system based on TDP-43 dimerization recognition of DNA targets according to claim 1, characterized in that: After the first step of expressing the FokT fusion protein in cells, only when TDP-43 binds to the target DNA site in a dimer state, the two FokI cleavage domains approach to form a dimer, thereby activating its enzymatic cleavage function and generating detectable double-stranded DNA breaks at the binding site.

6. The genome editing and detection system based on TDP-43 dimerization to recognize DNA targets according to claim 1, characterized in that: The target cells in the second step are one or more of 293T, neurons, and iPSC-derived neural cells.

7. The genome editing and detection system based on TDP-43 dimerization recognition of DNA targets according to claim 1, characterized in that: In the second step, dsODN carries a known sequence tag and can be integrated into the genomic break at the FokI restriction site, thereby enabling subsequent high-throughput detection.

8. The genome editing and detection system based on TDP-43 dimerization to recognize DNA targets according to claim 1, characterized in that: The fourth step can analyze whether it is located in the gene promoter or enhancer region, or whether it overlaps with splicing regulation or disease-related exons.

9. The genome editing and detection system based on TDP-43 dimerization to recognize DNA targets according to claim 1, characterized in that: In the fifth step, the TDP-43 mutant is ΔNTD or the pathology-related mutation M337V.

10. Application of a genome editing and detection system based on TDP-43 dimerization to recognize DNA targets in the study of DNA functions of RNA-binding proteins.