CN111534578A - A method for high-throughput screening of target genes of eukaryotic cells interacting with pesticides - Google Patents

Abstract

The invention relates to a method for screening target genes of interaction between eukaryotic cells and pesticides in high throughput. Then the cell library is uniformly divided into two parts for culture, genomic DNA is collected and extracted, the sgRNA abundances of the two groups of cells are detected by a high-throughput sequencing method respectively, and target genes of interaction of eukaryotes and pesticides are screened according to the variation of the sgRNA abundances of the two groups of cells. The method has the greatest advantages that the target gene of the interaction between the eukaryotic cells and the pesticide is screened in the whole genome range, the target gene of the interaction between the eukaryotic cells and the pesticide can be screened to the greatest extent, the cost is low, the efficiency is high, and the range is wide. Has great significance for screening target genes of interaction between eukaryotic cells and pesticides.

Description

A method for high-throughput screening of target genes of eukaryotic cells interacting with pesticides

technical field

The invention belongs to the technical field of gene editing and high-throughput sequencing, and relates to a method for high-throughput screening of target genes for interaction between eukaryotic cells and pesticides.

Background technique

my country is a big agricultural country, and agriculture has always been the lifeblood of the national economy. The use of pesticides brings convenience to modern agricultural production and greatly improves the quality and output of agricultural products. However, due to the widespread application of pesticides, pesticide residues contaminate soil and water sources, destroy the diversity of agricultural ecosystems, and seriously threaten human health. As a result, people have paid more attention to non-toxic and harmless biological pesticides. However, more and more practices have found that people always think that low-toxic biological pesticides are toxic to a variety of non-target organisms in the environment. . On the other hand, the phenomenon of insect resistance to pesticides has become increasingly prominent. This has not only become a major obstacle to pest control, but at the same time, the increase in pest resistance has led to increased use of pesticides, and the containment of insect resistance has become a major issue in the development of agriculture and forestry in recent years.

In addition, pesticides also pose a huge threat to the production of important economic insects such as silkworms and bees. As for the sericulture industry, pesticide poisoning causes a large amount of production reduction every year, which severely limits the development of the sericulture industry. The cultivation of silkworm varieties with high pesticide resistance needs to be solved urgently.

Gene editing is an important genetic manipulation technology for the study of functional genomes. It has been developed for four generations, including megaendonuclease, zinc finger nuclease, transcription factor activator-like nuclease, CRISPR and so on. Currently the most widely used is the CRISPR/Cas9 system. The CRISPR/Cas9 system is a gene editing technology derived from the acquired immune system of bacteria. Gene editing has been successfully achieved in species including human, mouse, Drosophila, Arabidopsis, and rice. The CRISPR/Cas9 system mainly includes two parts, one part is the non-specific endonuclease Cas9, and the other part is the guide RNA. Because of the simple design and construction, low cost and high editing efficiency, the current application of CRISPR/Cas9 system is not limited to single gene editing, and its application has been extended to multiple gene editing and even whole genome editing. Genome editing mediated by the CRISPR/Cas9 system has been achieved in humans, mice, Drosophila and other species. The genome-wide editing mediated by the CRISPR/Cas9 system has achieved important results in the research fields of drug target gene screening, tumorigenesis, and immune response.

It is of great significance to comprehensively use CRISPR/Cas9-mediated whole-genome editing methods to explore the target genes of eukaryotic interactions with pesticides.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a method for high-throughput screening of target genes for interaction between eukaryotic cells and pesticides.

To achieve the above object, the present invention provides the following technical solutions:

A method for high-throughput screening of target genes interacting between eukaryotic cells and pesticides, the specific steps are as follows:

(1) Construction of a eukaryotic CRISPR/Cas genome-wide editing vector library delivered by a transgenic system;

(2) stably integrating the vector library constructed in step (1) into the eukaryotic cell genome to obtain a eukaryotic CRISPR/Cas whole genome editing cell library;

(3) The cell library constructed in step (2) is evenly divided into two parts, one of which is cultured with a medium containing pesticides as an experimental group, and the other is cultured with a medium without pesticides as a control group, and then simultaneously Two groups of cells were collected, and genomic DNA was extracted respectively;

(4) Using the entire genome extracted in step (3) as a template, design primers to amplify the sgRNA fragments of each group of cells, perform high-throughput sequencing, count the abundance of sgRNA, and screen eukaryotic cells interacting with pesticide targets Gene, the screening standard of target gene is p-value<0.05.

As one of the preferred technical solutions, the eukaryotic organisms are silkworms, fruit flies, humans, etc., and the pesticides are organophosphorus pesticides, alkaloid pesticides, pyrethroid pesticides, biogenic pesticides, and the like.

As one of the preferred technical solutions, the concrete method of step (1) is:

(1-1) Design target sites, design about 6 target sites for each gene, and synthesize a single-stranded oligonucleotide library containing the target sites by means of DNA chips;

(1-2) The obtained single-stranded oligonucleotide library is cloned into a transgenic vector to construct a gene editing vector library.

As one of the further preferred technical solutions, according to the law of Cas action, the editing sites of all protein-encoding genes are designed at the eukaryotic whole genome level, and the targeting sites have the following rules:

5'-NNNNNNNNNNNNNNNNNN-NGG-3', the designed sgRNA sequence is consistent with its target site sequence on the genome, and has the following rules: 5'-NNNNNNNNNNNNNNNNNNN-3'; according to the above rules, all eukaryotic encoded proteins are designed target site of the gene.

As one of the further preferred technical solutions, the synthetic single-stranded oligonucleotide library is cloned into the vector pB-CRISPR by using the bridging PCR technology and the restriction enzyme ligation technology to construct the gene editing vector library pB-CRISPR-library. As one of the preferred technical solutions, the specific method of step (2) is: stably integrate the vector library constructed in step (1) into the eukaryotic cell genome, and the integration methods include lentivirus system, transposon system, site Specific nuclease system, etc., to obtain a CRISPR/Cas genome-wide editing cell library.

As one of the preferred technical solutions, the specific method of step (3) is as follows: the cell library constructed in step (2) is evenly divided into two parts, one of which is cultured with a medium containing pesticides until the number of cells is reduced to 5 %, the other was cultured with pesticide-free medium for the same time as a control group, and then two groups of cells were collected at the same time, and the genomic DNA was extracted respectively.

As one of the preferred technical solutions, in step (4), compared with the control group, the genes enriched or depleted of sgRNA in the experimental group are candidate target genes for the interaction between eukaryotic cells and pesticides.

The beneficial effects of the present invention are:

In the present invention, a eukaryotic CRISPR/Cas whole genome editing vector library is first constructed, and then the vector library is stably integrated into a eukaryotic cell line to construct a eukaryotic whole genome editing mutant cell library. Then the cell library was taken and divided into two parts, one of which was cultured with pesticide-containing medium until the number of cells decreased to 5%, and the other was cultured with pesticide-free medium for the same time as a control group. The cells of the two groups of experiments were collected and extracted at the same time, and their sgRNA abundances were detected by high-throughput sequencing, respectively, and the target genes of eukaryotic cells interacting with pesticides were screened. The biggest advantage of the present invention is to screen the target genes of the interaction between eukaryotic cells and pesticides in the whole genome without preconditions. Compared with the traditional researcher method of target genes interacting between eukaryotic cells and pesticides, the present invention can screen the target genes interacting between eukaryotic cells and pesticides to the greatest extent, with low cost, high efficiency and wide range. . It is of great significance for the screening of target genes of eukaryotic cells and pesticide interactions and the study of eukaryotic resistance to pesticides.

Description of drawings

In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for description:

Figure 1 shows the top 20 genes with significant enrichment of gRNAs screened by abamectin. The known drug resistance-related genes P450, etc. were screened, and some possible resistance genes were also screened (dark font) , a gene with transporter activity;

Figure 2 shows that among the top 20 genes with significantly enriched gRNA abundance screened by azadirachtin, some possible resistance genes (dark font) and genes with transporter activity were screened;

Figure 3 shows the top 20 genes with significant enrichment of gRNAs screened by rotenone. Known drug resistance-related genes such as P450 were screened, and some possible resistance genes (dark font) were also screened. gene for carrier activity;

Figure 4 shows the top 20 genes whose gRNA abundance was significantly depleted by the three pesticides of biological origin. There is a large overlap, which may indicate that cells respond to different pesticides with a common mechanism. ;

Figure 5 shows the number of genes with significant differences in gRNA abundance screened for the three biologically derived pesticides and the number of genes that overlap with several known drug resistance-related gene families.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Pesticide is an important plant protection method in modern agricultural production, which is of great significance to crop production. However, pesticides also bring many negative effects. Pesticide pollution is a major threat to ecological balance and human health. It is of great value to screen target genes that interact with eukaryotic cells and pesticides.

The following specific experimental methods that are not specified are implemented in accordance with recognized experimental methods and conditions, for example, according to the instructions provided by the reagent and consumable manufacturers, or according to the classic experimental book "Molecular Cloning Experiment Guide" (Third Edition, J. Mbruck waiting) to complete the experiment.

Example:

The silkworm embryonic cell line (The Bombyx mori embryonic cell line, BmE) used in this example is a cell line commonly used in biological experiments (PMID: 17570024).

1. Construct the BmE whole genome editing cell library of silkworm embryo cell line.

1) Construct a piggyBac transposon system-mediated silkworm CRISPR/Cas9 whole genome editing vector library. Most genes have been designed with 6 target sites and 6 gene editing vectors have been constructed; a total of 94,000 genes have been designed and constructed Edit vector.

2) Transfecting the silkworm whole genome editing mutant library constructed in step 1) and the piggyBac transposase expression vector A3-helper (the nucleotide sequence of which is as SEQ ID NO.1) in a molar ratio of 1:1 to the silkworm embryonic cell line BmE, Transfection methods include, but are not limited to, lipofection, electroporation, and the like. Then, the complete medium containing Zeocin was used to screen for 2 months, and the whole genome editing cell library of silkworm BmE cells was constructed. The complete cell culture medium contains 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and penicillin-streptomycin (Penicillin-Streptomycin, 200,000 units/liter, Thermo Fisher Scientific) ) of Grace Insect Medium (Grace's InsectMedium, Thermo Fisher Scientific). The culture condition was 27°C, Zeocin was purchased from Thermo Fisher, and the working concentration was 200 μg/ml.

2. Divide the whole genome editing cell library of silkworm BmE cells constructed in step 1 into two equal parts, one of which contains pesticides {the pesticides used in this experiment have three biological sources: Abamectin, working concentration 20 μM; rotenone, working concentration 500 nM; azadirachtin, working concentration 30 μM. Correspondingly, control cells were also cultured in three groups of medium until the number of cells decreased to 5%, another group was cultured in medium without pesticides for the same time as a control group, and then the two groups of cells were collected simultaneously.

3. Extract genomic DNA from the two groups of cells collected in step 2 respectively.

4. According to the piggyBac transposon system-mediated silkworm CRISPR/Cas9 whole genome editing vector library constructed in step 1, design a pair of primer pairs for amplifying sgRNA fragments.

Forward primer>gD-F, 5-NNNNNNNNNNNNTAAATCACGCTTTCAATA, N represents base A, T, G or C, as shown in SEQ ID NO.2;

Reverse primer>gD-R, 5-NNNNNNNNNNNNCGACTCGGTGCCACTTT, N represents base A, T, G or C, as shown in SEQ ID NO.3.

5. Using the two sets of genomes extracted in step 3 as templates, use the primer pair gD-F/gD-R described in step 4 to amplify the sgRNA fragments, and then perform high-throughput sequencing. All the genomes obtained in step 3 should be used for PCR amplification.

6. By analyzing the high-throughput sequencing data in step 5, the abundance of sgRNA in the two groups of cells was counted, and the target genes that interacted with silkworm cells and pesticides were analyzed and screened. Compared with the control group, the experimental group sgRNA was enriched or depleted. The gene is a candidate target gene for interaction between silkworm cells and pesticides.

8. Screening results of the interaction target genes between silkworm cells and pesticides

1) Among the top 20 genes with significant enrichment in gRNA abundance screened by abamectin, known drug resistance-related genes such as P450 were screened, and some possible resistance genes were also screened (dark font) , genes with transporter activity, see Figure 1.

2) Among the top 20 genes with significantly enriched gRNA abundance screened by azadirachtin, some possible resistance genes (dark fonts) and genes with transporter activity were screened, as shown in Figure 2.

3) Among the top 20 genes with significant enrichment in gRNA abundance screened by rotenone, the known drug resistance-related genes P450, etc. were screened, and some possible resistance genes (dark font) were also screened. The gene for vector activity is shown in Figure 3.

4) The top 20 genes that were significantly depleted in gRNA abundance by the three pesticides of biological origin screened by us have a large overlap, which may indicate that cells respond to different pesticides with a common mechanism, see Fig. 4.

5) The number of genes with significantly different gRNA abundances screened from the three biologically derived pesticides and the known gene families of several major categories of drug resistance-related genes are shown in Figure 5.

9. The above experiments show that the use of CRISPR-mediated whole genome editing cell library can effectively screen out the target genes that interact with eukaryotic cells and pesticides.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should Various changes may be made in details without departing from the scope of the invention as defined by the claims.

sequence listing

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Claims (6)

1. A method for screening target genes of interaction between eukaryotic cells and pesticides in high throughput is characterized by comprising the following specific steps:

(1) constructing a eukaryotic CRISPR/Cas whole genome editing vector library delivered by a transgenic system;

(2) stably integrating the vector library constructed in the step (1) to a eukaryotic cell genome to obtain a eukaryotic CRISPR/Cas whole genome editing cell library;

(3) uniformly dividing the cell library constructed in the step (2) into two parts, wherein one part is cultured by using a culture medium containing pesticide to serve as an experimental group, the other part is cultured by using a culture medium without pesticide to serve as a control group, then simultaneously collecting two groups of cells, and respectively extracting genome DNA;

(4) and (3) taking all the genomes extracted in the step (3) as templates, designing primers to amplify the sgRNA fragments of each group of cells, performing high-throughput sequencing, counting the abundance of the sgRNA, and screening target genes interacted between eukaryotic cells and pesticides, wherein the screening standard of the target genes is p-value < 0.05.

2. The method of claim 1, wherein the eukaryote is silkworm, fruit fly, human, and the pesticide is organophosphorus pesticide, alkaloid pesticide, pyrethroid pesticide, biogenic pesticide, or the like.

3. The method according to claim 1, wherein the specific method of step (1) is:

(1-1) designing targeting sites, designing about 6 targeting sites for each gene, and synthesizing a single-stranded oligonucleotide library containing the targeting sites by means of a DNA chip;

(1-2) cloning the obtained single-stranded oligonucleotide library to a transgenic vector to construct a gene editing vector library.

4. The method according to claim 1, wherein the specific method of step (2) is: stably integrating the vector library constructed in the step (1) into a eukaryotic cell genome in an integration mode including a lentivirus system, a transposon system, a site-specific nuclease system and the like to obtain a CRISPR/Cas whole genome editing cell library.

5. The method according to claim 1, wherein the specific method of step (3) is: uniformly dividing the cell library constructed in the step (2) into two parts, wherein one part is cultured by using a culture medium containing pesticide until the number of cells is reduced to 5%, the other part is cultured by using a culture medium without pesticide for the same time to be used as a control group, then simultaneously collecting two groups of cells, and respectively extracting genome DNA.

6. The method according to claim 1, wherein in the step (4), the sgRNA enriched or depleted genes in the experimental group are candidate target genes for interaction between the eukaryotic cells and the pesticide compared with the control group.

Images