Evaluation method for gene drift risk of gene editing fish based on eDNA

Abstract

The invention aims to provide an eDNA technology-based evaluation method for gene drift risk of gene editing fish and application of the eDNA technology-based evaluation method. According to the method, water samples are continuously collected in a culture water body of gene editing fishes and a drainage path of the culture water body, whether edited target gene drift exists in the culture and wastewater discharge process or not can be detected, a new technical means is provided for environmental safety assessment of gene editing animals, and the problem of risk monitoring of gene drift in a water environment is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosafety assessment, and specifically relates to an assessment method for gene drift risk of gene-edited fish based on eDNA. Background Art

[0002] Genome editing technology is a gene manipulation technology developed in recent years that can modify the genome at a specific point. With the rapid development of animal gene editing technology, the industrialization of gene-edited animals has also shown good application prospects. However, in recent years, there have been reports that some transgenic plants have undergone gene drift in the external environment, which makes the environmental safety of gene-edited animals an issue that needs to be explored urgently.

[0003] Gene drift refers to the spread of exogenous genes or edited genes of genetically modified animals to wild populations through contact, reproduction, lactation and other behaviors in breeding, as well as through intestinal microbial flora, which may have unforeseen effects on the balance of ecosystems and biodiversity. Therefore, detecting whether edited genes will drift in the environment is an important part of assessing the environmental safety of gene-edited animals.

[0004] Environmental DNA (eDNA) refers to the DNA left by organisms in the environment, usually from shed tissues, mucus, secretions, blood and corpses of organisms. eDNA technology is an ecosystem detection technology that has received widespread attention in recent years. It is usually used to monitor aquatic biodiversity and has broad application prospects. Previously, Qi Chuanxiang (2018) and others collected soil microbial DNA in the surrounding environment of aquaculture and conducted detection and analysis to detect whether the MSTN gene of pigs after editing may undergo gene drift through feces and other pathways. However, there have been no relevant research reports on the use of eDNA technology to monitor the environmental safety of aquatic gene-edited animals. In actual application, there are still many challenges in monitoring eDNA in aquaculture water environment. Unlike the soil environment, the content of fish secretions and shed tissues in the water environment is relatively small, and a large amount of solid particles in the turbid water of aquaculture can easily clog the pore size of the filter membrane, which may affect the enrichment of eDNA to a certain extent. Therefore, it is urgent to develop appropriate monitoring methods for monitoring the risk of gene drift of gene-edited animals in the water environment. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application for evaluating the risk of gene drift in gene-edited fish based on eDNA technology.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose includes the following specific steps:

[0007] 1) Collection of water environment samples:

[0008] Several sampling points were set up on the drainage path of the gene-edited fish breeding pond, and bottom water samples were collected using water samplers. The water environment samples collected at each sampling point were stored independently;

[0009] 2) Pretreatment of water environment samples:

[0010] The water environment sample collected in step 1) is first filtered through a silk sieve to remove large solid particles, and then filtered using a filter membrane. The filter membrane after filtration is placed in a refrigerated centrifuge tube and stored in liquid nitrogen for later use;

[0011] The sieve silk is 200 μm sieve silk, and the filter membrane is 5 μm polycarbonate membrane with a diameter of 47 mm;

[0012] 3) Enrichment of eDNA:

[0013] Cut the filter paper stored in step 2) into pieces, add ethylenediaminetetraacetic acid (EDTA) solution, sodium dodecyl sulfate (SDS) solution, and proteinase K solution in sequence, mix well, and treat in a 56°C warm bath;

[0014] The concentration of the ethylenediaminetetraacetic acid (EDTA) solution is 0.11M, and the pH is 8.0; the concentration of the sodium dodecyl sulfate (SDS) solution is 10%, and the pH is 5.75; the concentration of the proteinase K solution is 10 mg / mL, and the volume ratio of the EDTA solution: SDS solution: proteinase K solution is 600: 66: 6.6;

[0015] 4) Extraction and amplification of eDNA:

[0016] The eDNA on the filter paper was extracted as a PCR template, and PCR amplification was performed using universal primers for detecting marine fish and PCR primers for detecting the target site of the target gene. The amplified products were sequenced to determine whether Cynoglossus semilaevis was present in the sample and whether the edited target gene fragment was present.

[0017] Among them, the universal primers for marine fish detection have the following sequence information:

[0018] Upstream primer MiFish-F: 5′-GTCGGTAAAACTCGTGCCAGC-3′ (SEQ ID NO: 1);

[0019] Downstream primer MiFish-R: 5′-CATAGTGGGGTATCTAATCCCAGTTTG-3′ (SEQ ID NO: 2).

[0020] The primer pair used to detect the target gene Dmrt1 has the following sequence information:

[0021] Upstream primer Dmrt1TALEN-S: 5′-CGGGCAAAGGGAGAAGG-3′ (SEQ ID NO: 3);

[0022] Downstream primer Dmrt1 TALEN-A: 5′-AAAAACATCTCCTGAGGGCTAA-3′ (SEQ ID NO: 4).

[0023] The present invention continuously collects water samples from the breeding water bodies and drainage pathways of gene-edited fish, and can detect whether there is any drift of the edited target gene during the breeding and wastewater discharge process, providing a new technical means for the environmental safety assessment of gene-edited animals and solving the problem of gene drift risk monitoring in the water environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Drainage path diagram of gene-edited fish breeding pond;

[0025] Figure 2 : Actual photos of sampling points of gene editing drainage path;

[0026] Figure 3 :eDNA sample PCR agarose gel electrophoresis results;

[0027] Figure 4 : eDNA sample MiFish amplified PCR product sequencing NCBI comparison results. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below in conjunction with the embodiments.

[0029] Example 1: Monitoring of Gene Drift Risk in Male Cynoglossus Semilaevis Dmrt1 Homozygous Mutant Fish

[0030] Taking a group of male semi-smooth tongue sole Dmrt1 homozygous mutant fish raised at the Weizhuo Aquaculture Base in Tangshan City, Hebei Province (39°37′N, 118°10′E) as an example, the homozygous mutant male fish and several ordinary male and female fish were raised together in a 20*5 cement-casting culture pond. The specific steps include:

[0031] 1. Collection of water environment samples:

[0032] Five sampling points were set up on the drainage path of the gene-edited fish breeding pond. The specific location and environment are as follows: Figure 1 , Figure 2As shown, they are A: in the breeding pond where male and female common fish and knockout fish are mixed, B: the drainage outlet on the side of the breeding pond, C: the total drainage outlet outside the breeding pond, D: 5m outside the drainage outlet of the breeding pond, and E: 20m outside the drainage outlet of the breeding pond. 3L of bottom water samples were collected from each sampling point using a water sampler. The water environment samples collected from each sampling point were stored independently, and a total of 15 water samples were collected.

[0033] 2. Pretreatment of water environment samples:

[0034] The 15 water environment samples collected in step 1 were filtered on site with 200 μm silk sieves to remove large solid particles; the water environment samples after coarse filtration were placed in a filtration cup and filtered using a vacuum pump and a 5 μm polycarbonate membrane (diameter 47 mm); the filtered membrane was placed in a frozen centrifuge tube and stored in liquid nitrogen. After each filter paper was filtered, the equipment was cleaned with a large amount of pure water to prevent cross contamination.

[0035] 3. eDNA Enrichment:

[0036] Cut the 15 filter papers in step 2 into pieces, add 0.11 M ethylenediaminetetraacetic acid (EDTA) solution with a pH of 8.0, 10% sodium dodecyl sulfate (SDS) solution with a pH of 5.75, and 10 mg / mL proteinase K solution (the usage ratio of the three solutions is EDTA: SDS: proteinase K = 600 μl: 66 μl: 6.6 μl) in sequence, mix well, centrifuge briefly, and incubate at 56°C for 24 h.

[0037] 4. Extraction and amplification of eDNA:

[0038] 1. Extract DNA as PCR template:

[0039] (1) After the incubation, the sample was briefly centrifuged and placed in a 2 ml centrifuge tube. 220 μl of lysis buffer (5 M NaCl, pH=8.86; 10% CTAB) was added. After vortexing, the sample was incubated at 56°C for 10 min.

[0040] (2) Add an equal volume of chloroform to the lysed sample, vortex and centrifuge at 13,800 rpm for 10 min, and collect 800 μl of the supernatant.

[0041] (3) Using a DNA extraction kit (ZYMO RESEARCH), add twice the volume of DNA Binding Buffer to the supernatant, mix by pipetting, and place in an adsorption column. Centrifuge at 13,800 rpm for 2 min until all the mixed solution passes through the adsorption column. Add 400 μL Wash Buffer again, centrifuge at 13,800 rpm for 1 min, discard the filtrate, and add 200 μL Wash Buffer and repeat the operation.

[0042] (4) After the alcohol has completely evaporated, place the upper centrifuge column in a new 1.5 ml centrifuge tube, add 15 μL of 10 mM Tris-Cl (pH = 8), let stand for 1 min, and centrifuge at 13,800 rpm for 2 min; remove the DNA from the centrifuge tube, re-pass the column to maximize DNA acquisition, and centrifuge again for 2 min.

[0043] (5) Take 1.5 μL of sample for NanoDrop mass measurement, label the sample and store it at -80°C.

[0044] 2. PCR detection of fish residues in genomic eDNA

[0045] Using the marine fish universal primer MiFish, it can detect more than 230 species of subtropical marine fish.

[0046] The PCR primer sequences are

[0047] Upstream primer MiFish-F: 5′-GTCGGTAAAACTCGTGCCAGC-3′;

[0048] Downstream primer MiFish-R: 5′-CATAGTGGGGTATCTAATCCCAGTTTG-3′.

[0049] The reaction system and conditions are as follows:

[0050]

[0051] The reaction conditions are as follows:

[0052]

[0053] 3. PCR detection of site-directed mutation of Dmrt1 gene in genomic eDNA

[0054] For the detection primers of dmrt1 gene mutation fish, the PCR primer sequences are

[0055] Upstream primer Dmrt1TALEN-S: 5′-CGGGCAAAGGGAGAAGG-3′;

[0056] Downstream primer Dmrt1TALEN-A: 5′-AAAAACATCTCCTGAGGGCTAA-3′.

[0057] The reaction system and conditions are as follows:

[0058]

[0059] The reaction conditions are as follows:

[0060]

[0061] 4. Sequencing of PCR products

[0062] The PCR products were run on 1.5% agarose gel electrophoresis. Figure 3 The results showed that a 250bp band was detected after MiFish was amplified from eDNA at the five sampling points, proving that marine fish residues existed in the environment. However, no band was found after Dmrt1 site-directed mutagenesis primers were amplified from the five sampling points. Since the copy number of the dmrt1 gene was too low to be detected, there was no possibility of gene drift. Furthermore, the MiFish amplified PCR product was sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for sequencing. The results are as follows: Figure 4 The sequence was compared with the genome sequence of Semilaevis semilaevis by NCBI and found to be 100% similar to that of Semilaevis semilaevis.

[0063] It can be seen that this method can detect the DNA of specific fish residues from the environment and test whether there is site-specific mutation of the edited gene in the environment. Combined with the analysis and monitoring of whether there is a risk of drift of specific genes of gene-edited fish in the environment, it has great promotion and application value in the field of environmental safety and gene-edited animal safety testing.

Claims

1. A method for assessing the risk of gene drift in gene-edited fish based on eDNA technology, characterized in that: The method comprises the following steps: 1) Collection of water environment samples: Several sampling points were set up on the drainage path of the gene-edited fish breeding pond, and bottom water samples were collected using water samplers. The water environment samples collected at each sampling point were stored independently; 2) Pretreatment of water environment samples: The water environment sample collected in step 1) is first filtered through a silk sieve to remove large solid particles, and then filtered using a filter membrane. The filter membrane after filtration is placed in a refrigerated centrifuge tube and stored in liquid nitrogen for later use; 3) Enrichment of eDNA: Cut the filter paper stored in step 2) into pieces, add ethylenediaminetetraacetic acid solution, sodium dodecyl sulfate solution, and proteinase K solution in sequence, mix well, and heat-treat at 56°C; 4) Extraction and amplification of eDNA: The eDNA on the filter paper was extracted as a PCR template, and PCR amplification was performed using universal primers for detecting marine fish and PCR primers for detecting the target site of the target gene. The amplified products were sequenced to determine whether Cynoglossus semilaevis was present in the sample and whether the edited target gene fragment was present.

2. The method according to claim 1, characterized in that The sieve silk in 2) is 200 μm sieve silk.

3. The method according to claim 1, characterized in that The filter membrane in 2) is a 5 μm polycarbonate membrane with a diameter of 47 mm.

4. The method according to claim 1, characterized in that The concentration of the ethylenediaminetetraacetic acid solution in 3) is 0.11 M, and the pH is 8.0; the concentration of the sodium dodecyl sulfate solution is 10%, and the pH is 5.75; and the concentration of the proteinase K solution is 10 mg / mL.

5. The method according to claim 4, characterized in that The concentration of the ethylenediaminetetraacetic acid solution: sodium dodecyl sulfate solution: proteinase K solution is 600:66:6.

6.

6. The method according to claim 1, characterized in that The universal primers for detecting marine fish in 4) have an upstream primer sequence of SEQ ID NO: 1 and a downstream primer sequence of SEQ ID NO:

2.

7. The method according to claim 1, characterized in that The PCR primers for detecting the target site of the target gene in 4) have an upstream primer sequence of SEQ ID NO: 3 and a downstream primer sequence of SEQ ID NO: 4.

Images