CN112813106A

CN112813106A - Preparation method of zebra fish with LDLR gene deletion

Info

Publication number: CN112813106A
Application number: CN202110043825.1A
Authority: CN
Inventors: 汪利平
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-01-13
Filing date: 2021-01-13
Publication date: 2021-05-18

Abstract

The invention constructs LDLR gene deletion zebra fish by using CRISPR/Cas9 technology, and the specific steps comprise: 1) determining an LDLR target site according to the LDLR gene sequence; 2) preparing gRNA; 3) transcribing Cas9mR NA in vitro; 4) cas9mRNA and gRNA are mixed and injected into zebra fish embryos at the single cell stage; 5) f is to be₀Feeding the embryo to sexual maturity; 6) outcrossing with wild adult fish, screening F₀(ii) a 7) Selection of F producing efficient mutations₀Selfing and screening F₁(ii) a 8) From F₁Selecting female fish and male fish with the same mutation from the generation mutants, and hybridizing to obtain F₂Generation; 9) screening of L DLR Gene knockout homozygotesNamely the zebra fish with the LDLR gene deletion of stable inheritance. The LDLR gene deletion zebra fish constructed by the invention can be stably inherited and can be used for researching vascular embolism.

Description

Preparation method of zebra fish with LDLR gene deletion

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to preparation of zebra fish with LDLR gene deletion.

Background

The death rate of cardiovascular diseases is very high, and the death rate of cardiovascular diseases becomes the first important cause of death of human diseases, wherein the most important pathological factor is atherosclerosis, which is promoted by multiple factors, generally, the atherosclerosis is infiltrated by immune cells on the inner wall of a large and medium arterial blood vessel, lipid substances such as cholesterol, lipoid and the like are adhered to the blood vessel wall, tissue hyperplasia such as lipid stripes, lipid nuclei, calcareous deposits, fibers and the like is generated, and finally hematoma rupture, massive hemorrhage, thrombus and the like are formed, so that a series of fat metabolism disorders, neurovascular dysfunction, lumen stenosis, artery elasticity reduction and the like are caused.

LDLR is one of low-density lipoprotein receptors and hepatocyte surface receptors, and binds Apoe to remove lipoprotein particles in blood, and also interacts with lipoprotein B on LDL particles to remove LDL from blood, which is very important for removing cholesterol and triglyceride-rich lipoprotein particles in blood.

The zebra fish is a third-large vertebrate model except rats and mice, is fertilized in vitro, developed in vitro, transparent in embryo, visible in development process and large in one-time egg laying quantity, and is a good animal model for researching cardiovascular diseases.

Disclosure of Invention

The invention aims to construct an LDLR gene deleted zebra fish by using a CRISPR/Cas9 system.

The preparation process of the invention is as follows:

the DNA sequence of the target site of the invention is as follows: 5'-GGTAGCTGGAAGTGTGATGG-3' are provided.

The invention provides a gene targeting kit, which comprises two Oligo sequences with the sequence of 5'-TAGGTAGCTGGAAGTGTGATGG-3', 5'-AAACCCATCACACTTCCAGCTA-3', and can be used for silencing LDLR gene expression.

The invention provides a gene knockout method, which comprises the following steps: the Oligo fragment is used for recognizing a target site of a target gene, binding with Cas9 and recognizing a PAM sequence at the target site, guiding nuclease to bind to the target site of the target gene, starting shearing to form a DSB gap, then connecting a cell with a repair mechanism through a non-homologous end to repair a double chain of the target gene, causing frameshift mutation, and finally knocking out the target gene.

Further, the target point is one or more than one.

Further, the invention provides a preparation method of the zebra fish with the LDLR gene deletion, which comprises the following steps:

1. determining an LDLR target site according to the LDLR gene sequence;

2. designing an Oligo sequence according to the LDLR target site;

3. constructing a gRNA in vitro transcription vector;

4, PCR to obtain a gRNA in vitro transcription template;

5. carrying out in-vitro transcription on the template obtained in the step 4 to obtain gRNA;

6. preparing an in vitro transcription template of Cas9 mRNA;

7. transcribing Cas9mRNA in vitro;

8. adding a polyA sequence, and recovering Cas9 mRNA;

9. cas9mRNA and gRNA are mixed and injected into zebra fish embryos at the single cell stage;

10. f is to be₀Feeding the embryo to sexual maturity;

11. outcrossing with wild adult fish, screening F₀；

12. Selection of F producing efficient mutations₀Selfing and screening F₁；

13. Selecting female fish and male fish with the same mutation from the F1 generation mutants, and hybridizing to obtain F2 generation;

14. screening LDLR gene knockout homozygote to obtain the stably inherited LDLR gene deletion zebra fish.

The invention has the advantages that:

the zebra fish with the LDLR gene deletion constructed by the invention is the first case at home and abroad.

The LDLR gene deletion zebra fish constructed by the invention can be stably inherited and can be used for researching vascular embolism.

Drawings

FIG. 1: electrophoretic picture of clone framework pT7-gRNA-Bbs I

FIG. 2: PCR identification of bacterial liquid

FIG. 3: gRNA in vitro transcription template

FIG. 4: in vitro transcription of Cas9mRNA

FIG. 5: PCR and enzyme digestion identification mutant

FIG. 6: comparing CYP1B1 gene-deleted zebra fish with wild type sequence

Detailed Description

The present invention is further illustrated in detail by the following examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1: preparation of animal models of the invention

1. Laboratory animal

Wild type zebra fish (strain TU) were bred according to a standardized protocol with water temperature of 28.5 ℃ and light/dark cycle of 14h/10h, adult zebra fish were spawned and embryo collected and bred in E3 hatching fluid, and embryo and larval stages of development were expressed in hours of fertilization (hpf) or days of fertilization (dpf).

CRISPR/Cas9 Gene knockout target site design

Inquiring a zebra fish LDLR gene sequence on NCBI, designing an LDLR target site on http:// zifit.partners.org/ZiFiT/CSquarere 9 nucleic.aspx according to a CRISPR/Cas9 knockout principle, wherein the target site comprises 20 bases, and the selection standard of the target site is as follows: 5 '-GG- (N) 18-NGG-3'; wherein the 5 'GG dinucleotide is part of the T7 promoter, and the 3' end of the target site is NGG.

3. Construction of gRNA in vitro transcription vector

3.1 the gRNA cloning backbone pT7-gRNA-Bbs I was obtained by digesting pT7-gRNA with Bbs I and recovering the gel (see FIG. 1), the digestion system is shown in the following Table 1:

table 1 enzyme system:

3.2 ordering two oligos according to target site, oligo1 sequence 5'-TAGGTAGCTGGAAGTGTGATGG-3', oligo2 sequence 5'-AAACCCATCACACTTCCAGCTA-3';

3.3 using ddH₂O respectively dissolving the oligos into 10 mu M solution, and annealing to obtain small sticky end fragments, wherein the annealing procedure is shown in Table 2;

TABLE 2 annealing procedure

3.4 the annealed fragment is connected with the recovered gRNA cloning skeleton pT7-gRNA-Bbs I, transformed, picked and cloned, the RV-M and Oligo2 are used as primers to carry out the PCR identification of bacterial liquid (annealing at 58 ℃, extending for 30sec, 30 cycles), the target band is about 130bp (shown in figure 2), the positive clone is picked and sent to sequencing, and clone glycerol with correct sequence is selected for preserving bacteria, improving grains, and RV-M sequence: 5'-AGCGGATAACAATTTCACACAGGA-3', see Table 3 for the linkage system;

TABLE 3 connection System

4. Preparation of gRNA

4.1 using T7-cr fwd and tracr rev primer pair, using constructed gRNA in vitro transcription vector as template, obtaining gRNA in vitro transcription template (annealing at 58 ℃, extending for 30sec, 40cycle, 40 mul system) by using high fidelity enzyme PCR, taking 1 mul PCR product for electrophoresis, directly recovering PCR product after confirming single band (125bp) (see figure 3), for subsequent experiment, T7-cr fwd sequence: 5'-GAAATTAATACGACTCACTATA-3', tracr rev sequence: 5'-AAAAAAAGCACCGACTCGGTGCCAC-3', respectively;

4.2 in vitro transcription of gRNAs

The gRNA in vitro transcription system is shown in Table 4;

TABLE 4 in vitro transcription of mRNA reaction System

4.3 recovery of gRNA

4.3.1 RNase-free water was used to dilute the gRNA transcript to 300. mu.l, 330. mu.l of absolute ethanol was added;

4.3.2 adding the solution into a recovery column, centrifuging for 15s at 10000 g/min;

4.3.3 adding 700 mul of miRNA Wash Solution I, and centrifuging for 5-10 s;

4.3.4 adding 500 μ l of Wash Solution II, centrifuging for 5-10s, and repeating once;

4.3.5 discarding the liquid in the collecting pipe, centrifuging for 1min, and removing the residual liquid;

4.3.6 adding a proper amount of RNase-free water preheated at 95 ℃, centrifuging at the maximum rotating speed for 20-30s, and collecting a gRNA solution;

5. preparation of Cas9mRNA

5.1 preparation of in vitro transcription template for Cas9 mRNA: the pSP6-2sNLS-spCas9 vector (37 ℃, 4h or more) was linearized by Xba I single digestion; taking a small amount of electrophoresis to confirm that linearization is complete, and directly recovering a linearization product;

5.2 in vitro transcription of Cas9mRNA, the mRNA in vitro transcription system is shown in Table 5;

TABLE 5 Cas9mRNA in vitro transcription System

5.3 addition of polyA sequence, mRNA recovered (FIG. 4) can be used for microinjection;

TABLE 6 mRNA plus polyA reaction System

6. Preparation F₀Zebra fish substitute

6.1 mixing Cas9mRNA and gRNA, injecting into a zebra fish embryo of a single cell, and simultaneously taking the same batch of uninjected embryos as a control, wherein Cas9mRNA 300-500pg and gRNA 25-200 pg;

6.2 taking the embryo with normal phenotype after 2-4dpf injection, extracting genome DNA, and detecting the mutation efficiency of the target site by PCR and T7E1 enzyme digestion (figure 5);

6.3 recovering the uncut band, TA cloning and sequencing, and detecting the mutation type;

6.4 selection of F for injection in the same batch with higher mutation efficiency and higher survival rate₀Feeding the embryo to sexual maturity;

6.5 carrying out outcrossing with wild adult fish, mixing 3-5F 1 embryos with 1dpf into a group to extract genome DNA;

6.6 detecting the mutation condition of the target site by PCR and enzyme digestion;

6.7 recovering the band which is not cut, TA clone sequencing and determining the mutation type;

6.8 selection of F producing potent mutations₀Mating fish and breeding in large quantities F₁。

7. Screening for F carrying the mutation at the target site₁Adult fish

7.1 mixing F₁Raising to be large enough until tail fins are suitable to be cut;

7.2F₁cutting tail fin and extracting gene group of adult fish, carrying out gene detection one by PCR and enzyme digestion, and screening out F₁Heterozygote;

7.3 recovering the band which is not cut open and TA clone, sequencing and determining the mutation type;

8. selecting female fish and male fish with the same mutation from the F1 generation mutation body weight, hybridizing to obtain F2 generation, culturing at 28.5 ℃, taking partial embryos at 4dpf, independently extracting genome DNA of each embryo, and taking primer pairs with the following primer sequences:

9. the efficiency of identifying homozygous mutations by PCR banding analysis was further identified by sequencing (figure 5).

Claims

The preparation method of the zebra fish with the LDLR gene deletion is characterized by comprising the following steps:

1) determining an LDLR target site according to the LDLR gene sequence;

2) designing an Oligo sequence according to the LDLR target site;

3) constructing a gRNA in vitro transcription vector;

4) PCR is carried out to obtain a gRNA in vitro transcription template;

5) carrying out in-vitro transcription on the template obtained in the step 4 to obtain gRNA;

6) preparing an in vitro transcription template of Cas9 mRNA;

7) in vitro transcription of Cas9mRN_A；

8) Adding a polyA sequence, and recovering Cas9 mRNA;

9) cas9mRNA and gRNA are mixed and injected into zebra fish embryos at the single cell stage;

10) f is to be₀Feeding the embryo to sexual maturity;

11) outcrossing with wild adult fish, screening F₀；

12) Selection of F producing efficient mutations₀Selfing and screening F₁；

13) From F₁Selecting female fish and male fish with the same mutation from the generation mutants, and hybridizing to obtain F₂Generation;

14) screening LDLR gene knockout homozygote to obtain the stably inherited LDLR gene deletion zebra fish.
2. The use of the LDLR gene deleted zebrafish of claim 1, wherein the LDLR gene deleted zebrafish is used for the study of vascular embolism.