CN113897360A - Eef1b2 gene knockout zebra fish epilepsy model and application thereof - Google Patents

Abstract

According to the invention, through a CRISPONT technology, a 5bp fragment is inserted into a second exon of a zebra fish eef1b2 gene, and a 3bp fragment is deleted at the same time, so that a eef1b2 gene-deleted zebra fish is bred, and thus a zebra fish epilepsy model is constructed. The invention uses four sgRNAs to target the same gene, so that the editing efficiency can be improved to the maximum extent, and the gene editing can be carried out more effectively. The zebra fish epilepsy model provided by the invention lays a good foundation for further developing the relation research of EEF1B2 mutation and epilepsy pathogenesis and screening antiepileptic drugs.

Description

Eef1b2 gene knockout zebra fish epilepsy model and application thereof

Technical Field

The invention relates to the technical field of gene knockout, in particular to an eef1b2 gene knockout zebra fish epilepsy model and application thereof.

Background

CRISPR (clustered, regular internationalized, short palindromic repeats) is an immunological mechanism from bacteria to degrade invading viral DNA or other foreign DNA. In bacteria and archaea, CRISPR systems are divided into 3 types, wherein the types I and III need multiple CRISPR-associated proteins (Cas proteins) to play a role together, while the type II system only needs one Cas protein, which provides convenience for wide application. At present, the CRISPR/Cas9 system is most widely used. Researchers found that targeting the same gene with multiple sgrnas could maximize editing efficiency, and named this approach as crisp technology.

In the results of sequencing by complete exome (WES) of the epileptics in the previous period, the gene EEF1B2 which possibly causes epileptic diseases is screened, and no report of EEF1B2 mutant epileptic zebra fish line is found through literature inquiry. Zebrafish, as a vertebrate, have a complex nervous system, are similar in genetic structure to humans, and share approximately 70% of genes with humans, and about 84% of genes related to human diseases are known to be expressed in zebrafish. Compared with rodents such as mice and the like, the zebra fish is simple in genetic operation and more suitable for preparing an epilepsy model. And the zebra fish eef1b2 gene is more conservative in evolution, and the research finds that the eef1b2 has particularly high expression level in the early embryo stage of the zebra fish. Therefore, the invention aims to establish EEF1B2 gene knockout zebra fish epilepsy model by using CRISPONT technology, and the zebra fish can be used for researching the relation between EEF1B2 mutation and epilepsy pathogenesis and screening subsequent antiepileptic drugs.

Disclosure of Invention

The invention provides an EEF1B2 gene knockout zebra fish epilepsy model and application thereof, and lays a foundation for further researching the relation between EEF1B2 mutation and an epilepsy pathogenesis and screening antiepileptic drugs.

The invention adopts the following technical scheme:

the invention provides a zebra fish epilepsy model, which is constructed by knocking out a zebra fish eef1b2 gene by utilizing sgRNA combination, wherein the sgRNA combination sequence is shown as SEQ ID No. 1-SEQ ID No.4, the zebra fish eef1b2 gene is knocked out by inserting a 5bp fragment into a second exon of a zebra fish eef1b2 gene and deleting a 3bp fragment, and the sequences are shown as SEQ ID No.7 and SEQ ID No. 8.

The invention provides an application of sgRNA combination in construction of a zebra fish epilepsy model, which is characterized in that the sgRNA combination is used for knocking out a zebra fish eef1b2 gene to construct the zebra fish epilepsy model, the sgRNA combination sequence is shown in SEQ ID No. 1-SEQ ID No.4, the knocked-out zebra fish eef1b2 gene is obtained by inserting a 5bp fragment into a second exon of a zebra fish eef1b2 gene and deleting a 3bp fragment, and the sequence is shown in SEQ ID No.7 and SEQ ID No. 8.

The third aspect of the invention provides application of the zebra fish epilepsy model in screening antiepileptic drugs.

The fourth aspect of the invention provides a method for constructing a zebra fish epilepsy model, which comprises the following steps:

1) and sgRNA combination of the targeted CRISPONT gene and screening of detection primers: the sgRNA combined sequence is shown in SEQ ID NO.1 to SEQ ID NO.4, and the detection primer sequence is shown in SEQ ID NO.5 to SEQ ID NO. 6;

2) injecting a mixture of 4 sgrnas and Cas9 proteins into fertilized eggs of zebrafish;

3) f0 mutant zebra fish screening: screening out effective embryo, and culturing to adult fish to obtain F0 generation mutant zebra fish;

4) obtaining generations F1 of heritable zebrafish mutants: hybridizing F0 generation mutant zebra fish with wild zebra fish to obtain F1 generation embryos, screening the embryos with mutation, culturing to adult fish, screening to obtain F1 generation of heritable zebra fish mutant, compared with wild type, inserting 5bp fragment into the second exon of eef1b2 gene and deleting 3bp fragment, the sequence is shown as SEQ ID NO.7 and SEQ ID NO. 8.

The invention has the beneficial effects that:

according to the invention, a sgRNA combination of a zebra fish epilepsy model is constructed by specifically knocking out the zebra fish eef1b2 gene through CRISPONT, a 5bp fragment is inserted into a second exon of the zebra fish eef1b2 gene and a 3bp fragment is deleted at the same time through CRISPONT technology, and the zebra fish with eef1b2 gene deletion is bred, so that the zebra fish epilepsy model is constructed.

The invention uses four sgRNAs to target the same gene, so that the editing efficiency can be improved to the maximum extent, and the gene editing can be carried out more effectively.

The zebra fish epilepsy model provided by the invention lays a good foundation for further developing the relation research of EEF1B2 mutation and epilepsy pathogenesis and screening antiepileptic drugs.

Drawings

Fig. 1 is a CRISPR/Cas9 knockout schematic.

FIG. 2 is a sequence position diagram of the target site on the genome of eef1b2 gene.

FIG. 3 is an electrophoresis detection diagram of 4 sgRNAs (M is a DNA Marker, and the numbers are 100bp, 200bp, 300bp, 400bp, 500bp, 700bp and 1000bp from bottom to top, and numbers 1, 2, 3 and 4 are sgRNA1, sgRNA2, sgRNA3 and sgRNA4 respectively).

FIG. 4 shows 4 sgRNA activity-verifying electropherograms (M is a DNA Marker, which is 100bp, 200bp, 300bp, 400bp, 500bp, 700bp, and 1000bp from bottom to top in sequence; Nos. 1, 2, and 3 are embryos after single-target injection of sgRNA1, Nos. 4, 5, and 6 are embryos after single-target injection of sgRNA2, Nos. 7, 8, and 9 are embryos after single-target injection of sgRNA3, Nos. 10, 11, and 12 are embryos after single-target injection of sgRNA4, and WT is a wild type).

FIG. 5 shows eef1b 2F 0 generation genotype identification electrophoretograms (M is DNA Marker, which is 100bp, 200bp, 300bp, 400bp, 500bp, 700bp, 1000bp from bottom to top in sequence; numbers 1-10 are F0 generation fish tail serial numbers, WT is wild type).

FIG. 6 shows eef1b 2F 1 generation genotype identification electrophoretograms (M is DNA Marker, which is 100bp, 200bp, 300bp, 400bp, 500bp, 700bp, 1000bp from bottom to top in sequence; numbers 1-15 are F1 generation fish tail serial numbers, WT is wild type).

FIG. 7 is a comparison of the sequencing peaks of the eef1b 2F 1 generation wild type and mutant.

Detailed Description

The present invention will be described in further detail with reference to the following examples and the accompanying drawings. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

1) Design CRISPONT gene knockout target site and detection primer

The genomic DNA sequence of The zebrafish eef1b2 Gene (Gene number: Gene ID:335370) was queried on The National Center for Biotechnology Information (NCBI), and The target site of The eef1b2 Gene was designed on The website The ZFIN (https:// ZFIN. org /) according to The CRISPR/Cas9 knock-out principle (FIG. 1). The selection of the target must follow this criterion: 5 '-GG- (N) 18-NGG-3'. The GG dinucleotide at the 5 'end is part of the T7 promoter, and the target site can be designed without limitation, but the NGG at the 3' end of the target site must be ensured. The target site must be selected within the domain of the gene to ensure that the insertion or deletion of the base of the target site can affect the entire domain of the eef1b2 gene to alter gene expression. The target site of the gene to be knocked out is located in the second exon of eef1b2 gene (FIG. 2), and the sequence of sg RNA is shown in Table 1.

Table 1sgRNA sequences

Primers were designed in Primer Premier 3.0 software (shown in table 2) from a genomic region of about 200bp upstream and downstream of the crispan target site of the eef1b2 gene.

TABLE 2 primer sequences

2) sgRNA synthesis and quality control

And (3) carrying out PCR experiment by using the designed primers to detect whether the designed sgRNA sequence is wrong. After confirming that the sgRNA was correct, the designed sgRNA was sent to a commercial company (south kyo jinsley biotechnology limited, hereinafter abbreviated as south kyo jinsley) for synthesis.

The four sgRNAs were centrifuged at 14000rpm for 10min to precipitate the RNA dry powder. Then, 15. mu.L of RNase-free double distilled water was added to dissolve the RNA dry powder. And performing quality detection on the four dissolved sgRNAs.

First, 1 μ L of sgRNA solution was taken and the concentration was measured on an ultraviolet spectrophotometer, and the concentration of each sgRNA and OD260/280 data were recorded. Secondly, 1 mu L of sgRNA solution is mixed with loading buffer for agarose gel electrophoresis, and whether the sgRNA is a single band is detected. Microinjection experiments can be performed if the sgRNA concentration is high (at least higher than 600 ng/. mu.l) and the electrophoretic band is a uniform band. The results are shown in fig. 3, which indicates that 4 sgRNA bands are single bands, contain no impurities, have high concentration, and can be used for microinjection experiments.

3) Activity validation of Single sgRNA

Before formal targeting, whether the designed sgRNA can be edited effectively needs to be tested. Thus, single sgRNA activity validation was performed. Cas9 protein (Nanjing Kisrei, Z03389-50) was complexed with 4 different sgRNAs as per the system in Table 3, giving a final concentration of Cas9 protein of 250 ng/. mu.l and a final concentration of sgRNAs of 100 ng/. mu.l. About 1nL of Cas9 protein and sgRNA cocktail was injected into fertilized eggs at one cell stage. The injected fertilized eggs were placed in E3 water and incubated at 28 ℃. Embryo phenotype is observed under a body type microscope, and the embryo which normally develops is screened for target site mutation analysis.

TABLE 3sgRNA and cas9 protein complexation System

4) Sanger sequencing to detect effectiveness of sgRNA

After microinjection is carried out on zebra fish embryos, partial normally developed early embryos are selected, whether the eef1b2 gene of the early embryos has mutation or not is detected, whether the selected target site has the effect or not is confirmed in advance, and whether microinjection operation is standard or not is determined.

a. Extraction of zebra fish genome

After 24 hours of fertilization (24hpf) of zebra fish embryos, wild type 1 tube (as a control) and injected experimental group embryos (3 tubes for each experimental group) are collected in 1.5mL Ep tubes (5 embryos per tube), and lysate is added to extract genomic DNA.

b. PCR amplification of target sequences

After extraction of genomic DNA, the PCR reaction system of Table 4 was used to amplify the DNA fragment of interest using the primer sequences of Table 2.

TABLE 4PCR reaction System

Shaking and mixing evenly, centrifuging, and carrying out amplification reaction on a PCR instrument. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, (denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and elongation at 72 ℃ for 30s) for 35 cycles, and then at 72 ℃ for 8 min. After the reaction is finished, centrifuging the PCR product, taking 2 microliter of sample to sample on 1.3% agarose gel for electrophoresis, and detecting whether the size of the PCR product is correct or not.

c. And if the PCR product is correct, sending the PCR product to perform Sanger sequencing, wherein the result shows that the band is single as shown in FIG. 4, and the PCR product can be sent to a commercial company (biological engineering (Shanghai) corporation) to perform Sanger sequencing, the insertion or deletion information is preliminarily obtained from a sequencing peak diagram, the sgRNA knockout efficiency is obtained after comparison through a tide website, and formal injection is performed after the sgRNA is determined to be effective, wherein the result is shown in Table 5.

TABLE 5 Single sgRNA knockout efficiency

	sgRNA1	sgRNA2	sgRNA3	sgRNA4
					Knock out efficiency	72％	78％	50％	40％

4) Microinjection of zebrafish embryos

Within 30min after fertilization, embryos were pipetted into a microinjection petri dish made of agarose.

Before microinjection, the Cas9 protein and 4 different sgrnas were mixed well to prepare a mixture, so that the final concentration of Cas9 protein was 250ng/μ l and the final concentration of each sgRNA was 100ng/μ l. About 1nL of Cas9 protein and sgRNA cocktail was injected into fertilized eggs at one cell stage. The injected fertilized eggs were placed in E3 water and incubated at 28 ℃. Embryo phenotype is observed under a body type microscope, and the embryo which normally develops is screened for target site mutation analysis.

Selecting 3 tubes of embryos (5 per tube) to detect the target knocking-out efficiency, wherein the detection steps are the same as the above steps, and culturing the rest embryos to two months old after the knocking-out is determined to be effective.

5) F0 generation mutant zebra fish screening

a. Extraction of zebra fish genome

After the embryo is cultured for two months, part of tail fin tissues of adult zebra fish are collected in a 1.5mL centrifuge tube, and lysate is added into an EP tube to extract genome DNA.

b. PCR amplification of target sequences

After extraction of genomic DNA, the PCR reaction system of Table 4 was used to amplify the DNA fragment of interest using the primer sequences of Table 2. The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, (denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and elongation at 72 ℃ for 30s) for 35 cycles, and then at 72 ℃ for 8 min. After the reaction is finished, centrifuging the PCR product, taking 2 microliter of sample to sample on 1.3% agarose gel for electrophoresis, and detecting whether the size of the PCR product is correct or not.

c. If the PCR product is correct, the PCR product is sent to perform Sanger sequencing, the experimental result is shown in FIG. 5, the result shows that the band is single, the PCR product can be sent to a commercial company (Biotechnology engineering (Shanghai) GmbH) to perform Sanger sequencing, the information of insertion or deletion is obtained from a sequencing peak diagram, and F0 zebra fish carrying the mutation is screened.

6) Generation F1 to obtain heritable zebrafish mutants

The zebra fish mutant F0 generation was confirmed by the previous series of screens, and then the F0 generation mutant was respectively crossed with wild zebra fish to obtain F1 generation embryos, which were cultured at 28 ℃ and the survival rate of F1 generation was observed at the early stage.

The zebrafish mutant was bred for up to 2-3 months for F1 generations if the presence of a mutation was detected from F1 generation embryos. And then, respectively shearing tails of each F1-generation adult zebra fish, and screening F1-generation mutants (the specific method is described in step 5), wherein the experimental result is shown in FIG. 6, and the result shows that the stripe is single and can be sent to a commercial company (biological engineering (Shanghai) GmbH) for Sanger sequencing. According to the analysis of the Sanger sequencing result of the screened F1 generation mutant, as shown in FIG. 7, according to the sequencing peak chart, the eef1b2 mutant inserts a 5bp fragment on the second exon and simultaneously deletes a 3bp fragment compared with the wild type sequence, thereby causing frame shift mutation. The red boxes indicate the sequence of sgRNA4, the blue square frame being the deleted 3 bp. Inserted 5bp fragment sequence: TTTGG, missing 3bp fragment sequence: and (4) CTT.

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Claims (4)

1. A zebra fish epilepsy model is characterized in that a zebra fish eef1b2 gene is knocked out by utilizing sgRNA combination, the sgRNA combination sequence is shown as SEQ ID No. 1-SEQ ID No.4, the zebra fish eef1b2 knocked-out gene is obtained by inserting a 5bp fragment into a second exon of a zebra fish eef1b2 gene and deleting a 3bp fragment, and the sequences of the zebra fish epilepsy model are shown as SEQ ID No.7 and SEQ ID No. 8.

2. An application of sgRNA combination in construction of a zebra fish epilepsy model is characterized in that the zebra fish epilepsy model is constructed by knocking out a zebra fish eef1b2 gene by the sgRNA combination, the sgRNA combination sequence is shown in SEQ ID No. 1-SEQ ID No.4, the zebra fish eef1b2 knocked-out gene is obtained by inserting a 5bp fragment into a second exon of a zebra fish eef1b2 gene and deleting a 3bp fragment, and the sequences are shown in SEQ ID No.7 and SEQ ID No. 8.

3. The use of the zebrafish epilepsy model of claim 1 for screening antiepileptic drugs.

4. A construction method of a zebra fish epilepsy model is characterized by comprising the following steps:

1) and sgRNA combination of the targeted CRISPONT gene and screening of detection primers: the sgRNA combined sequence is shown in SEQ ID NO.1 to SEQ ID NO.4, and the detection primer sequence is shown in SEQ ID NO.5 to SEQ ID NO. 6;

2) injecting a mixture of 4 sgrnas and Cas9 proteins into fertilized eggs of zebrafish;

3) f0 mutant zebra fish screening: screening out effective embryo, and culturing to adult fish to obtain F0 generation mutant zebra fish;

4) obtaining generations F1 of heritable zebrafish mutants: hybridizing F0 generation mutant zebra fish with wild zebra fish to obtain F1 generation embryos, screening the embryos with mutation, culturing to adult fish, screening to obtain F1 generation of heritable zebra fish mutant, compared with wild type, inserting 5bp fragment into the second exon of eef1b2 gene and deleting 3bp fragment, the sequence is shown as SEQ ID NO.7 and SEQ ID NO. 8.

Images