WO2019127116A1 - Cell clone for cell phenotypic studies, screening method therefor, and application thereof - Google Patents

Abstract

Provided are a cell clone for cell phenotypic studies, a screening method therefor, and an application thereof. The screening method for the cell clone for cell phenotypic studies is mainly applicable when searching for a cell population of a certain cell phenotype in cells having not been subjected to experimental treatment. A gRNA barcode vector acts both as a barcode and a retrieval tool, thereby simplifying a searching and screening process. A first reporter gene of a gRNA-sensor search vector itself is not normally expressed, and signals only in response to action with a specific gRNA barcode and a nuclease for binding to the gRNA barcode, thereby allowing qualification in the search process. As long as a signal associated with the first reporter gene is present, the signal can be captured, thereby facilitating screening to obtain positive cell clones. The selected positive cells can be used in single studies, and are able to respond or generate phenotypes. The selected cells can also be widely used in studying cell phenotypes or in preparation of reagents for studying cell phenotypes.

Description

Cell clone for cell phenotype research and screening method and application thereof Technical field

The invention relates to the field of molecular biology, in particular to a cell clone for cell phenotype research and a screening method and application thereof.

Background technique

In organisms, cells have different phenotypes, and it is important to study the underlying causes of different phenotypes. Taking cancer cells as an example, in cancer patients, tumor cell populations will exhibit different phenotypes, such as tumor cells that can proliferate, some migrate, and some have drug resistance. These different phenotypes are progress in cancer research. There are many major causes of unpredictability in response to treatment, so understanding the basic biology of tumorigenesis is very important to improve treatment outcomes. The underlying cause of tumor cells with different phenotypes can be attributed to two mutually non-exclusive possibilities: one is that all tumor cells in the patient have the same intrinsic phenotypic potential, but there are many factor variables in the patient's body itself or during the medication. Random causes cause only some tumor cells to exhibit a certain phenotype; second, the patient's tumor cell population is heterogeneous in its genetic and epigenetic, and it is destined that only some cells exhibit corresponding phenotypes. Traditional methods for studying the causes of different phenotypes in different cell populations (such as tumor cells), such as the patent CN102203281A, are tools for making barcode clones that are lentiviral overexpression vectors, either by the 5'UTR of the reporter gene or A 10 base long random barcode is placed on the 3'UTR. In theory, there are 4 ¹⁰ combinations. The search tool used is a lentiviral shRNA interference vector, which specifically targets the reporter gene on the barcode tool. Fluorescent reporter cells are weakened by the target cells. However, traditional research methods generally have many problems, such as using shRNA interference carrier tools to search barcodes, reporting that gene detection signals are strongly weakened, while some cells undergo changes in external factors such as growth conditions during growth and passage. It is easy to make the fluorescence of itself weaken, and it is easy to mix negative cells, which is not conducive to observation and screening; and if you want to further eliminate the mixed cells, you should use doxycycline to re-screen and process the cells one more time. At the same time, it may affect the original characteristics of the cells; in addition, the retrieval process is a quantitative process, and it is difficult to retrieve those cells containing multiple copies of the barcode carrier.

Summary of the invention

Based on this, it is necessary to provide a cell clone for cell phenotype research which is easy to observe and screen and has high retrieval efficiency and reliable retrieval result, and a screening method and application thereof.

A screening method for cell clones for cell phenotype research, comprising the following steps:

Step 1: construct a gRNA barcode vector having an expression cassette for expressing gRNA used as a marker barcode, the gRNA barcode vector has various types, and different gRNA barcode vectors have different sequences of gRNA coding fragments, and various The gRNA barcode carrier constitutes a gRNA barcode carrier;

Step 2: transducing the gRNA barcode carrier library to the cells to be studied, and obtaining a cell population composed of a plurality of cells containing different gRNA coding fragments, and dividing the cell population into an experimental group and a reserved group;

Step 3: experimentally treating the cell population of the experimental group, screening the experimental group positive cells having a specific phenotype, and obtaining the sequence of the specific gRNA coding fragment corresponding to the specific phenotype in the positive cells of the experimental group. ;

Step 4: constructing a specific gRNA-sensor search vector for the sequence of the specific gRNA-encoding fragment obtained, the expression frame of the gRNA-sensor search vector containing the first reporter gene and inserted in the first reporter gene a left homology arm, a specific gRNA coding fragment, a PAM and a right homology arm, the left homology arm and the right homology arm being homologous to a partial sequence fragment of the first reporter gene;

Step 5: After the constructed specific gRNA-sensor search vector and the vector capable of expressing the nuclease for binding the gRNA barcode are used to transduce the cell population of the reserved group, the result is selected according to the expression of the first reporter gene. Positive cells, that is.

In one embodiment, the constructing the gRNA barcode vector is to insert the gRNA-encoding fragment into a vector having a suicide gene to replace the suicide gene.

In one embodiment, the gRNA-encoding fragment is further ligated to the protective sequence fragment and/or the cleavage site recognition fragment, respectively.

In one embodiment, the gRNA barcode has a length of 18-23 bp, preferably 20 bp.

In one embodiment, the protected sequence fragment is 40-50 bp in length, preferably 40 bp.

In one embodiment, the gRNA barcode vector further has an expression cassette for a second reporter gene.

In one embodiment, the second reporter gene and/or the first reporter gene is a fluorescent protein gene and/or a drug resistance gene.

In one embodiment, the gRNA barcode vector is a lentiviral vector.

In one embodiment, each cell to be studied is transduced with one or two gRNA barcode carriers.

In one embodiment, the step of expanding the resulting population of cells is further included prior to dividing the population of cells into an experimental group and a reserved group.

In one embodiment, the left homology arm and/or the right homology arm are 180-300 bp in length, preferably 200 bp.

In one embodiment, the vector capable of expressing a nuclease for binding to a gRNA barcode is a vector capable of expressing a Cas9 or saCas9 nuclease.

A cell clone obtained by screening a cell clone for cell phenotype research described in any of the above embodiments.

The above cell clones are used in the study of cell phenotypes or in the preparation of reagents for studying cell phenotypes.

The screening method for cell clones for cell phenotypic research of the present invention can be mainly used for searching for a cell phenotype (including a tendency to respond to an experimental treatment) from a cell that has not been subjected to experimental treatment, wherein gRNA The barcode carrier acts both as a barcode and as a retrieval tool, thus simplifying the process of searching and screening. Secondly, the first reporter gene of the gRNA-sensor search vector itself is not normally expressed, only with the specific gRNA barcode and The signal is signaled by binding to the nuclease of the gRNA barcode, so that the search process can be characterized; again, as long as there is a signal related to the first reporter gene, it can be captured, thereby facilitating screening to obtain positive cell clones.

Screened positive cells can be used in a single study to determine their ability to respond or phenotype to further determine the underlying cause of cells with different phenotypes, and can be widely used in studying cell phenotypes or in preparing for studying cell phenotypes. In the reagents.

DRAWINGS

Figure 1 is a schematic diagram showing the screening process of cell clones for cell phenotype research;

2 is a schematic diagram showing the structure and construction flow of a gRNA barcode carrier. In the figure, “NNNNNNNNNNNNNNNNNNNN” is a gRNA coding fragment of a gRNA barcode;

Figure 3 is a schematic diagram showing the structure of a gRNA-sensor search vector.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully hereinafter with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the understanding of the present disclosure will be more fully understood.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. The terminology used in the description of the present invention is for the purpose of describing particular embodiments and is not intended to limit the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

CRISPR/Cas9 (or variants thereof, such as saCas9) (Clustered Regularly Interspaced Short Palindromic Repeats) is a technique by which RNA directs Cas nuclease to perform specific DNA modification of a targeted gene. After gRNA binds to nucleases such as Cas9 and saCas9, the nuclease cleaves the DNA duplex by a PAM (Protospacer Adjacent Motif, NGG) sequence, resulting in DNA double-strand break. A screening method for cell clones for cell phenotypic research of an embodiment is mainly used for constructing a nuclease-assisted search for barcode cell clones, and screening for positive cell clones having a certain phenotype (including a tendency to respond to experimental treatment), Cell phenotypes were studied in depth. As shown in FIG. 1, the screening method of this embodiment includes the following steps:

Step 1: constructing a gRNA barcode vector having an expression cassette for expressing gRNA used as a marker barcode, the gRNA barcode vector is various, and different gRNA barcode vectors have different sequences of gRNA coding fragments, and various gRNAs The barcode carrier constitutes a library of gRNA barcode carriers.

The gRNA barcode is a fragment of a gRNA sequence having a specific sequence. By designing the gRNA barcode, each gRNA barcode vector and a cell subsequently transduced with the gRNA barcode vector have a specific gene signature. In a specific embodiment, the length of the gRNA barcode is 18-23 bp, preferably 20 bp (the sequence of the gRNA barcode of 20 bp length can be expressed as "NNNNNNNNNNNNNNNNNNNN"), that is, there are 4 ²⁰ kinds of gRNA barcodes theoretically having the length. Different combinations.

The gRNA barcode vector has at least one expression cassette for expressing a gRNA barcode, and the expression cassette is composed of a gRNA coding fragment of a gRNA barcode and a corresponding promoter, terminator and the like. In one embodiment, the gRNA-encoding fragment for expression of the gRNA barcode is further ligated with a protective sequence fragment and/or a restriction endonuclease fragment fragment, respectively. The protected sequence fragments and/or restriction endonuclease site fragments can be used as universal amplification primer fragments for different gRNA barcodes for PCR amplification and/or restriction endonuclease digestion. The length of the protected sequence fragment can be, but is not limited to, 40-50 bp, preferably 40 bp.

Further, in one embodiment, the empty vector used to construct the gRNA barcode vector has a suicide gene, such as a ccdB gene, into which the gRNA-encoding fragment of the gRNA barcode is inserted. The suicide gene can reduce the effect of the vector without the target gene, and the bacteria that subsequently select for transfection also use bacteria that cannot resist the suicide gene.

Still further, in one embodiment, the gRNA barcode vector further comprises another expression cassette for expression of a second reporter gene. The second reporter gene can be, but is not limited to, a fluorescent protein gene and/or a drug resistance gene. The design of the second reporter group facilitates the labeling and screening of vectors into which the sequence of interest is inserted.

As shown in Figure 2, in a specific embodiment, the gRNA barcode vector is a lentiviral vector, the promoter of the gRNA coding fragment containing the gRNA barcode is a U6 promoter, and the gRNA barcode of the gRNA barcode is inserted in U6 Downstream of the promoter, replacing the original suicide ccdB gene fragment; the promoter of another expression cassette is the PGK promoter, and the second reporter gene is a fluorescent protein gene such as mCherry and a drug resistance gene such as Puro, wherein the fluorescent protein gene can be used for expression. The fluorescently-labeled fluorescent protein, the drug-resistant gene makes the vector or the transfected cell resistant, and can be used for drug screening and enrichment of the desired expression vector or cell. The gRNA barcode vector can be, but is not limited to, a lentiviral vector, the promoter of the gRNA barcode can be U6 but not limited to the U6 promoter, and the promoter of the reporter gene expression cassette can be, but is not limited to, a PGK promoter.

Step 2: The gRNA barcode carrier library is transduced into the cells to be studied, and a cell population composed of a plurality of cells containing different gRNA coding fragments is obtained, and the cell population is divided into an experimental group and a reserved group.

Depending on the number of cells to be transformed, the amount of vector used in the gRNA barcode vector library is adjusted such that each cell to be studied is randomly transduced with one or two gRNA barcode carriers.

The step of expanding the obtained cell population is further included before dividing the cell population into the experimental group and the reserved group. The obtained experimental group cell group and the reserved group cell group have substantially the same number of cells labeled with each gRNA barcode, and have multiple copies, such as expanded culture, so that each gRNA barcode-labeled cell has about 100 sisters. Cells for easy analysis.

Step 3: Experimentally treating the cell population of the experimental group, screening the experimental group positive cells having a specific phenotype, and obtaining the sequence of the specific gRNA coding fragment corresponding to the specific phenotype in the positive cells of the experimental group.

The sequence of the specific gRNA-encoding fragment corresponding to the gRNA barcode in the experimental group positive cells can be separately read by, but not limited to, PCR or sequencing.

Step 4: construct a specific gRNA-sensor search vector for the sequence of the specific gRNA-encoding fragment obtained, and the gRNA-sensor search vector contains the first reporter gene and the left homolog inserted in the first reporter gene. The arm, the specific gRNA coding fragment, the PAM and the right homology arm, the left homologous arm and the right homology arm are homologous to a partial sequence fragment of the first reporter gene.

In one embodiment, the left homology arm and/or the right homology arm are 180-300 bp in length, preferably 200 bp.

As shown in FIG. 3, in a specific embodiment, the promoter of the gRNA-sensor search vector may be EF1A, and the first reporter gene may be a green fluorescent protein (EGFP) gene or the like.

Step 5: After the constructed specific gRNA-sensor search vector and the vector capable of expressing the nuclease for binding the gRNA barcode are used to transduce the cell population of the reserved group, the positive cells are selected according to the expression of the first reporter gene. That's it.

In one embodiment, the vector capable of expressing a nuclease for binding to a gRNA barcode is a vector capable of expressing a nuclease such as Cas9 or saCas9.

Taking the gRNA-sensor search vector shown in Figure 3 as an example, the nucleotide of the first reporter gene (such as EGFP gene) is divided into two parts, the upstream part and the downstream part, by the gRNA coding fragment + PAM of the specific gRNA barcode. There are left and right homology arms of about 200 bp in length. At this time, the first reporter gene cannot be expressed normally. When the specific gRNA-sensor search vector is transferred to the cell group of the reserved group together with the vector capable of expressing the nuclease for binding the gRNA barcode, the experimental group positive cells selected in the above step 3 have the same gRNA barcode. The gRNA expressed in the cell can be combined with the nuclease, and the target-specific gRNA-sensor search vector can be searched. At this time, the left and right homologous arms of the first reporter gene on the gRNA-sensor search vector are homologously recombined, first The reporter gene is normally expressed, such as the EGFP gene expressing EGFP, which emits a green fluorescent signal for screening.

Positive cell clones can be screened from the reserved group by signal changes in the expression of the first reporter gene from scratch, such as positive cell clones that can be screened for specific fluorescent signals by flow cytometry, and The positive cell clones with specific drug resistance are screened by drugs, and the selected positive cell clones are separately expanded and cultured, which can be used to further study the ability of response or phenotype formation, and the cells have different phenotypes. s reason. Therefore, the cell clones screened by the above screening methods can be used in the study of cell phenotypes or in the preparation of reagents for studying cell phenotypes.

The screening method of the cell clone for the above cell phenotypic study can be mainly used for searching a cell population of a certain cell phenotype (including a tendency to respond to an experimental treatment) from the untreated cells, wherein the gRNA barcode carrier It acts both as a barcode and as a search tool, thus simplifying the process of searching and screening. Secondly, the first reporter gene of the gRNA-sensor search vector itself is not normally expressed, only with specific gRNA barcodes and for binding to gRNA. The nuclease of the barcode signals the signal, so that the process can be characterized qualitatively; again, as long as there is a signal related to the first reporter gene, it can be captured, thereby facilitating screening to obtain positive cell clones.

The following is part of the specific examples.

The study found that mouse breast cancer cell TM40D-MB can be transferred to bone and form secondary tumors after implantation into mouse mammary fat pad. This cell line is a series of continuous cells in BALB/c mice by TM40 mother cells. Obtained from the transplant experiment. In order to observe the metastasis of cancer cells in real time in vivo, the Luc luciferase reporter gene was integrated into TM40D-MB (Li, Z., C. Schem, YHShi, D. Medina, and M. Zhang, Increased COX2 expression enhances tumor- In induced osteoclastic lesions in breast cancer bone metastasis. Clin Exp Metastasis, 2008. 25(4): p. 389-400.), TM40D-MB-Luc cells were formed. The ability of metastatic tumors to metastasize may be caused by random causes or by genetic factors. Studies have shown that cells derived from secondary tumors have stronger transfer ability, and it is never necessary to find out the root cause of their stronger metastatic potential. This method has never used the method of "nuclease-assisted search for barcode cloning". Sister cells that can form secondary tumors are obtained from transplanted mouse breast cancer cells TM40D-MB-Luc, and whether the formation of secondary tumors is random or whether these cells themselves have greater metastatic potential, the specific steps are as follows.

1. Construction of gRNA barcode carrier

As shown in Figure 2, there are two expression cassettes in the lentiviral vector, the expression cassette 1 is the U6 promoter-mediated expression of the gRNA-encoding fragment of gRNA; the expression cassette 2 drives the red fluorescent protein mCherry and puromycin Puro drug screening. The gene, red fluorescent protein is a visible marker, so that the gRNA barcode carrier can be observed after entering the cell, and puromycin can eliminate cells that do not contain the gRNA barcode vector.

The procedure for cloning a gRNA barcode fragment of a gRNA barcode into an empty barcode lentiviral vector is as follows:

1) synthesizing a single-stranded oligonucleotide gRNA coding fragment library by chip synthesis technology, each gRNA coding fragment is used to encode a gRNA barcode of 20 nt in length, and an additional amplification region is provided at both ends of the gRNA coding fragment, The amplification region comprises an enzyme cleavage site and a protective base (40 bases at both ends) for cloning the gRNA coding fragment;

Examples of sequences of partial gRNAs in the gRNA-encoding fragment library used are as follows:

gRNA1: TACGTCCCTGTGCAGCTGCA (SEQ ID NO. 1)

gRNA2: ACGTCCCTGTGCAGCTGCAA (SEQ ID NO. 2)

gRNA3: CGTCCCTGTGCAGCTGCAAG (SEQ ID NO. 3)

gRNA4: TACGTCCCTGTGCAGCTGCA (SEQ ID NO. 4)

gRNA5: GCACTACCAGAGCTAACTCA (SEQ ID NO. 5)

gRNA6: GGGGCCACTAGGGACAGGAT (SEQ ID NO. 6)

The cleavage site and the protection base sequence at both ends:

5' end sequence: TATATATCTTGTGGAAAGGACGAAACACCTTCGGCAGGTG (SEQ ID NO. 7)

3' end sequence: CACCTGCACCGGTTTTAGAGCTAGAAATAGCAAGTTAAAA (SEQ ID NO. 8)

2) Converting the single-stranded oligonucleotide gRNA-encoding fragment library into a double-stranded gRNA-encoding fragment library, using a single-stranded oligonucleotide gRNA-encoding fragment library as a template, PCR amplification using a high-fidelity enzyme, and purification using PAGE The method recovers an amplification product having a length of 100 bp;

The primers for PCR amplification are as follows:

F:TATATATCTTGTGGAAAGGACG (SEQ ID NO.9)

R: TTTTAACTTGCTATTTCTAGCTC (SEQ ID NO. 10)

3) Put the empty barcoded lentiviral vector of Figure 2 (the vector contains the ccdB suicide gene in the region of the gRNA coding fragment of the gRNA barcode to be cloned, the ccdB gene can be used to reduce the background of the empty bar code lentiviral vector) and the amplification product is put together The universal GoldenGate seamless digestion and ligation reaction was carried out, and then the ligated reaction product was transformed into E. coli competent state which could not resist the ccdB suicide gene to construct a final gRNA barcode vector library.

2. The gRNA barcode vector is packaged into a lentiviral vector, and the amount of virus is adjusted according to the number of TM40D-MB-Luc cells, so that each cell can randomly contain one or two gRNA barcodes. The red fluorescent protein mCherry was used to observe the virus transduction quality, and the puromycin Puro was used to screen the clones that obtained the gRNA barcode, and the cells containing the gRNA barcode library were expanded to be 6-7 generations so that each cell could contain about 100 sisters. cell.

3. The amplified cells are divided into two parts, one as an experimental group and one as a reserved group.

The experimental group was subjected to the following treatment: 2 × 10 ⁷ TM40D-MB-Luc cells were transplanted into 20 mouse breast tissues of BALB/c, and 1 × 10 ⁶ cells were transplanted per mouse. Within 4 weeks, the expression of luciferase Luc was observed using an optical microscopy imaging system, and the size of the tumor was monitored three times per week, and it was found that TM40D-MB-Luc cells formed a primary tumor of about 0.2 cm in the breast. After 4-8 weeks, half of the mice showed tumor metastasis to the bones, and the secondary tumors on the bones were monitored weekly to record the size and number of secondary tumors and the life span of the mice.

4. Read the gRNA barcode of the experimental group positive cells.

These secondary tumors are isolated from mice, and the cells that can form secondary tumors and have metastatic potential are the experimental group-positive cells to be studied. In theory, each secondary tumor cell is developed from a cell with metastatic potential. Each secondary tumor cell is isolated and expanded separately, genomic DNA is extracted, and the gRNA coding fragment is amplified by PCR amplification. The coding sequence of a specific gRNA barcode is confirmed by sequencing.

PCR amplification primers:

F: GAGGGCCTATTTCCCATGATTC (SEQ ID NO. 11)

R: AGCCTGCTTTTTTGTACAAACTTG (SEQ ID NO. 12)

5. Construction of gRNA-sensor search tool

The gRNA+PAM sequence fragment is cloned into the middle of the green fluorescent protein EGFP according to the sequence of the specific gRNA barcode, and specifically includes the following steps:

1) The entry cloning vector pDown-DR-EGFP-BsaI-ccdB-Cm-BsaI was digested with BsaI, and the target fragment pDown-DR-EGFP was recovered;

2) synthesizing gRNA+PAM forward and reverse two fragments, and cloning the gRNA+PAM sequence into pDown-DR-EGFP by annealing ligation to obtain pDown-DR-EGFP-gRNA-PAM vector;

Examples of two primers for gRNA+PAM positive and negative:

gRNA1-F: ctaaTACGTCCCTGTGCAGCTGCAAGG (SEQ ID NO. 13)

gRNA1-R: cgggCCTTGCAGCTGCACAGGGACGTA (SEQ ID NO. 14)

gRNA2-F: ctaaACGTCCCTGTGCAGCTGCAAGGG (SEQ ID NO. 15)

gRNA2-R: cgggCCCTTGCAGCTGCACAGGGACGT (SEQ ID NO. 16)

3) Using the Gateway technology, the promoter plasmid (such as pUp-EF1A), the reporter plasmid (pDown-DR-EGFP-gRNA-PAM) and the target plasmid (pRp) were subjected to LR reaction to obtain a specific gRNA-sensor search tool, as shown in the figure. 3.

6. Sister positive barcode cells of the experimental group positive cells are retrieved from the reserved group

The specific gRNA-sensor vector and the vector expressing Cas9 protein were separately transduced into the reserved cells, and the cell clones with green fluorescent signals were screened by flow cytometry (FACS). These clones have metastatic potential. Sisters were positive for barcode cells and some clones without fluorescent signal were screened as experimental negative controls.

7. These sister-positive barcode cells were separately expanded and the gRNA barcode sequences of these cells were further determined by PCR.

8. Each sister-positive barcode cell population was transplanted into the breast tissue of 20 BALB/c mice with the same number (1×10 ⁶ cells), and the size of the primary tumor and the secondary tumor were recorded. Data on the number and size of mice, the lifespan of mice, and the like, and cells with no metastatic ability (no fluorescence) were used as negative controls. These data were compared to experimental group data to analyze whether these cells have greater metastatic potential.

If only a small number of positive cells have metastatic ability or exhibit the same characteristics as the negative control, it can be determined that this metastatic ability is caused by random causes; if the primary tumor appears faster, larger, and transferred to the bone The more or faster the next generation of tumors, it can be considered that this metastatic ability is determined by genetic factors, and thus can carry out genotypic analysis of molecular biology such as whole genome sequencing, DNA methylation analysis.

The technical features of the above-described embodiments may be arbitrarily combined. For the sake of brevity of description, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be considered as the scope of this manual.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (10)

A screening method for cell clones for cell phenotype research, comprising the steps of: Step 1: construct a gRNA barcode vector having an expression cassette for expressing gRNA used as a marker barcode, the gRNA barcode vector has various types, and different gRNA barcode vectors have different sequences of gRNA coding fragments, and various The gRNA barcode carrier constitutes a gRNA barcode carrier; Step 2: transducing the gRNA barcode carrier library to the cells to be studied, and obtaining a cell population composed of a plurality of cells containing different gRNA coding fragments, and dividing the cell population into an experimental group and a reserved group; Step 3: experimentally treating the cell population of the experimental group, screening the experimental group positive cells having a specific phenotype, and obtaining the sequence of the specific gRNA coding fragment corresponding to the specific phenotype in the positive cells of the experimental group. ; Step 4: constructing a specific gRNA-sensor search vector for the sequence of the specific gRNA-encoding fragment obtained, the expression frame of the gRNA-sensor search vector containing the first reporter gene and inserted in the first reporter gene a left homology arm, a specific gRNA coding fragment, a PAM and a right homology arm, the left homology arm and the right homology arm being homologous to a partial sequence fragment of the first reporter gene; Step 5: After the constructed specific gRNA-sensor search vector and the vector capable of expressing the nuclease for binding the gRNA barcode are used to transduce the cell population of the reserved group, the result is selected according to the expression of the first reporter gene. Positive cells, that is. The method for screening a cell clone for cell phenotype research according to claim 1, wherein the gRNA barcode vector is constructed by inserting the gRNA coding fragment into a vector having a suicide gene to replace a suicide gene. The method for screening a cell clone for cell phenotype research according to claim 1, wherein a protective sequence fragment and/or an enzyme cleavage site recognition fragment are further ligated to both ends of the gRNA coding fragment. The method for screening a cell clone for cell phenotype research according to claim 3, wherein the gRNA barcode has a length of 18 to 23 bp; and/or the protection sequence fragment has a length of 40 to 50 bp. The method for screening a cell clone for cell phenotype research according to claim 1, wherein the gRNA barcode vector further has an expression cassette of a second reporter gene. A method for screening a cell clone for cell phenotype research according to any one of claims 1 to 5, wherein each cell to be studied is transduced with one or two gRNA barcode carriers. The method for screening a cell clone for cell phenotype research according to any one of claims 1 to 5, wherein the length of the left homology arm and/or the right homology arm is 180-300 bp . The method for screening a cell clone for cell phenotype research according to any one of claims 1 to 5, wherein the vector capable of expressing a nuclease for binding to a gRNA barcode is capable of expressing a Cas9 or saCas9 nucleic acid. The carrier of the enzyme. A cell clone obtained by screening a cell clone for cell phenotype research according to any one of claims 1 to 8. The use of a cell clone according to claim 9 for studying a cellular phenotype or for preparing a reagent for studying a cellular phenotype.

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