Method for efficiently constructing large-scale human membrane protein CRISPR (clustered regularly interspaced short palindromic repeats) plasmid library and application

Abstract

The invention discloses a method for efficiently constructing a large-scale human membrane protein CRISPR (clustered regularly interspaced short palindromic repeats) plasmid library and application, and relates to the technical field of gene editing. Specifically, the invention provides an efficient and accurate method for constructing CRISPR plasmids of a human membrane protein library containing 4318 membrane proteins, and the method is suitable for the fields of large-scale gene editing research, drug screening, virus receptor identification and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing, and specifically relates to a method and application for efficiently constructing a large-scale human membrane protein CRISPR plasmid library. Background Art

[0002] The CRISPR-Cas9 system is an efficient gene editing tool that can achieve precise editing of the genome by designing specific single guide RNA (sgRNA) sequences. The system has shown great application potential in the fields of gene function research, disease model construction, gene therapy, etc. The construction of a large-scale gene editing library is an important means to systematically study gene function. Through high-throughput screening, multiple genes can be systematically knocked out or edited at the cellular level to identify genes with specific functions.

[0003] Membrane proteins play an important role in biomedicine. They play an important role in cell signal transduction, material transport, immune response, etc. Many important physiological processes, such as nerve conduction and hormone signal transduction, rely on the function of membrane proteins. At the same time, membrane proteins also play a key role in many diseases. For example, abnormal expression of membrane proteins in cancer and dysfunction of membrane proteins in neurodegenerative diseases. Studying the function of membrane proteins helps to reveal the pathogenesis of diseases and provide new targets for disease treatment.

[0004] Viruses invade cells by binding to specific membrane proteins on the surface of their host cells. These membrane proteins are called viral receptors. By systematically screening and identifying these viral receptors, we can provide important targets for the prevention and treatment of viruses. By constructing a large-scale membrane protein library, we can use it for high-throughput screening of viral receptors.

[0005] Although existing technologies have made some progress in membrane protein research, the construction of large-scale membrane protein libraries still faces many challenges, such as incomplete membrane protein libraries, sgRNA design, efficient plasmid cloning and library homogeneity, etc. The present invention provides a systematic method, which significantly improves the construction efficiency and accuracy of large-scale membrane protein libraries by optimizing the construction of membrane protein lists, the design and synthesis of sgRNA sequences, the efficient cloning of plasmids and the construction of libraries, and is suitable for high-throughput screening and functional verification of viral receptors. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application for efficiently constructing a large-scale human membrane protein CR ISPR plasmid library in view of the deficiencies of the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme: As a first aspect, a method for efficiently constructing a large-scale human membrane protein CRISPR plasmid library is provided, comprising the following steps:

[0008] Constructing a list of target membrane proteins: Screen multiple target human membrane proteins from an existing gene database, construct a list (the embodiment of the present invention constructs a list of 4318 target human membrane proteins), and ensure the representativeness and diversity of the selected membrane proteins. Among them, screening multiple target human membrane proteins and constructing a list specifically includes: screening proteins with clear membrane localization, eliminating proteins without targeted sgRNA sequences, and constructing a list containing gene IDs and protein names.

[0009] Design specific sgRNA sequences: Use software tools such as CRISPR Design Tool, CRISPResso, and CHOPCHOP to design multiple specific sgRNA sequences for each target membrane protein. Specifically, 10 sgRNAs are designed for each target membrane protein. The top 10 sgRNA sequences are selected based on the high specificity and low off-target rate of sgRNA. If there are less than 10 sgRNA sequences, they are added repeatedly according to the scores.

[0010] Synthetic sgRNA sequence: Through synthetic biology technology, the designed sgRNA sequence is synthesized to ensure the accuracy and efficiency of the synthesis.

[0011] Cloning sgRNA sequence into CRISPR vector: Using Gibson Assembly method, the synthesized sgRNA sequence is cloned into CRISPR vector to construct CRISPR plasmid, thus improving the efficiency and success rate of cloning.

[0012] Construction of CRISPR plasmid library: The cloned CRISPR plasmids were mixed to construct a CRISPR plasmid library containing 4318 target membrane proteins, and the diversity and accuracy of the library were verified by sequencing.

[0013] As a second aspect, the present invention provides the application of the method for efficiently constructing a large-scale human membrane protein CRISPR plasmid library in large-scale gene editing research and / or drug screening and / or viral receptor identification.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] Accuracy: sgRNA sequences were designed using software tools such as CRISPR Design Tool, CRISPResso, and CHOPCHOP, ensuring high specificity and low off-target rate of sgRNA, improving the accuracy of gene editing, and 10 SgRNAs were designed for each target protein, with a large library capacity and targeting.

[0016] Diversity: The diversity of the CRISPR plasmid library is ensured through sequencing verification, covering 4318 target membrane proteins, which can fully cover the types of membrane proteins required for research. Compared with existing commercial knockout libraries, such as Addgene's GeCKOv2 Human CRISPR Knockout Pooled Library, which contains 19,050 gene knockouts but only more than 1,100 membrane proteins, the library of the present invention is richer and more comprehensive in covering membrane proteins.

[0017] Wide application: This CRISPR plasmid library is suitable for a variety of application scenarios such as large-scale gene editing research, drug screening, and viral receptor identification, and has broad application prospects and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a map of CRISPR vectors;

[0019] Figure 2 This is a graph showing the sequencing depth results of a large-scale human membrane protein CRISPR plasmid library;

[0020] Figure 3 This is a graph showing the uniformity of a large-scale human membrane protein CRISPR plasmid library. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The present invention provides an efficient and accurate method for constructing a CRISPR plasmid library containing 4318 target human membrane proteins. The method includes constructing a list of target membrane proteins, designing specific sgRNA sequences, synthesizing sgRNA sequences, cloning sgRNA sequences into CRISPR vectors, and constructing a CRISPR plasmid library. By using software tools such as CRISPR DesignTool, CRISPResso and CHOPCHOP to design sgRNA sequences, and using the Gibson Assembly method to clone sgRNA sequences, high specificity and low off-target rate of the library are ensured. The CRISPR plasmid library is suitable for large-scale gene editing research, drug screening and viral receptor identification, and has efficient, accurate and broad application prospects. The specific steps are as follows:

[0023] 1. Construct a list of target membrane proteins

[0024] Step 1.1: Screening of target membrane proteins

[0025] Data source: Known human membrane protein genes and related mass spectrometry data were screened from existing gene databases (such as NCBI, UniProt).

[0026] Screening criteria: Select proteins with clear membrane localization, including G protein-coupled receptors (GPCRs), ion channels, transporters, etc.

[0027] Software tools: Use bioinformatics tools (such as TMHMM, Phobius) to predict the transmembrane domain of membrane proteins to ensure that the selected protein is indeed expressed on the cell membrane.

[0028] Step 1.2: Build a list of membrane proteins

[0029] List content: Proteins without targeting sgRNA sequences were removed, and a list of 4318 target membrane proteins was constructed, each corresponding to a unique gene ID and protein name.

[0030] File format: Save the list as Excel or CSV file for later processing.

[0031] 2. Design specific sgRNA sequences

[0032] Step 2.1: Choose a design tool

[0033] Tool selection: Use software tools such as CRISPR Design Tool, CRISPResso, and CHOPCHOP to design sgRNA sequences.

[0034] Design criteria: Select sgRNA sequences with high specificity and low off-target rate, select the top 10 sg RNAs, and if there are less than 10 sgRNAs, add them to the library repeatedly according to the scores.

[0035] Step 2.2: Design sgRNA sequence

[0036] Input data: Input the gene sequences in the membrane protein list into the design tool.

[0037] Output data: Generate multiple sgRNA sequences for each target membrane protein, and select 10 best sgRNA sequences for each protein.

[0038] File format: Save the designed sgRNA sequence as a FASTA format file.

[0039] 3. Synthesize sgRNA sequence

[0040] Step 3.1: Select the synthesis service

[0041] Service provider selection: Choose Twist, a reliable synthetic biology service provider, to synthesize sgRNA sequences.

[0042] Order submission: Submit the designed sgRNA sequence file to the service provider to ensure the accuracy and efficiency of synthesis.

[0043] Step 3.2: Receiving the Synthetic Product

[0044] Product verification: After receiving the synthesized sgRNA sequence, sequencing verification is performed to ensure the correctness of the sequence.

[0045] Storage conditions: Store the synthesized sgRNA sequence in a -20°C refrigerator for future use.

[0046] 4. Cloning sgRNA sequences into CRISPR vectors

[0047] Step 4.1: Prepare CRISPR vector

[0048] Vector selection: Use the vector provided by Yuanjing Biotechnology. Figure 1 shown.

[0049] Vector treatment: Linearize the CRISPR vector using restriction endonucleases to ensure linearization efficiency.

[0050] Step 4.2: Cloning sgRNA sequences

[0051] Cloning method: The synthesized sgRNA sequence was cloned into the linearized CRISPR vector using the Gibson Assembly method.

[0052] Reaction conditions: Follow the instructions of the Gibson Assembly kit to ensure the optimization of reaction conditions.

[0053] Transformation and screening: The cloned products were transformed into E. coli and positive clones were screened by antibiotics.

[0054] Step 4.3: Verify cloning efficiency

[0055] Sequencing verification: Extract plasmids from positive clones and perform sequencing verification to ensure that the sgRNA sequence is correctly inserted into the CRISPR vector.

[0056] Documentation: Record the sequencing results of each clone to ensure the accuracy and completeness of the clone.

[0057] 5. Construction of CRISPR Plasmid Library

[0058] Step 5.1: Mix cloning plasmids

[0059] Plasmid extraction: Extract plasmids from verified positive clones to ensure the quality and concentration of the plasmids.

[0060] Mixed library: All extracted plasmids were mixed to construct a CRISP R plasmid library containing 4318 target membrane proteins.

[0061] Step 5.2: Verify library diversity

[0062] Sequencing verification: The constructed CRISPR plasmid library was subjected to high-throughput sequencing to verify the diversity of the library and ensure that the sgRNA sequence of each target membrane protein was evenly distributed in the library.

[0063] Data analysis: Bioinformatics tools were used to analyze sequencing data and assess the coverage and diversity of the libraries.

[0064] Table 1 shows the statistics of the sequencing data of the CRISPR plasmid library of the present invention. Figure 2 The sequencing depth of the CRISPR plasmid library is shown; the horizontal axis is the Log 2 value of the sequencing depth, and the vertical axis is the corresponding gRNA number. Figure 3 The uniformity of the CRISPR plasmid library is shown; wherein the horizontal axis is the sequencing depth, the vertical axis is the cumulative frequency of gRNA, the lower left value is the number of gRNAs corresponding to 10% pct, the middle left value is the number of gRNAs corresponding to 90% pct, and the Skew Ratio is the ratio of the two (i.e., the ratio of the 90% quantile to the 10% quantile).

[0065] Table 1 Statistics of large-scale human membrane protein CRISPR plasmid library sequencing data

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[0068] The above embodiments are only used to illustrate the ideas and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, any equivalent changes or modifications made based on the principles and design ideas disclosed by the present invention are within the protection scope of the present invention.

Claims

1. A method for efficiently constructing a large-scale human membrane protein CRISPR plasmid library, characterized in that: The following steps are involved: Screen multiple target human membrane proteins from existing databases to build a list; For each target human membrane protein, multiple sgRNA sequences are designed, the sgRNA sequences are synthesized, and the synthesized sgRNA sequences are cloned into CRISPR vectors to construct CRISPR plasmids; the CRISPR plasmids are mixed to construct a CRISPR plasmid library.

2. The method according to claim 1, characterized in that The screening of multiple target human membrane proteins and construction of a list specifically includes: screening proteins with clear membrane localization, eliminating proteins without targeted sgRNA sequences, and constructing a list containing gene IDs and protein names.

3. The method according to claim 1, characterized in that The design of multiple sgRNA sequences specifically includes: designing sgRNA sequences using CRISPR Design Tool, CRISPResso and CHOPCHOP software, selecting the top 10 sgRNA sequences based on high specificity and low off-target rate, and repeatedly adding sgRNA sequences according to the scores if there are less than 10 sgRNA sequences.

4. The method according to claim 1, characterized in that: The cloning of the synthesized sgRNA sequence into the CRISPR vector and the construction of the CRISPR plasmid are achieved by the Gibson Assembly method.

5. The method according to claim 1, characterized in that The diversity of the constructed CRISPR plasmid library was verified by sequencing.

6. An application of the method for efficiently constructing a large-scale human membrane protein CRISPR plasmid library as described in claim 1 in large-scale gene editing research and / or drug screening and / or viral receptor identification.

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