WO2018219093A1 - Method for constructing glrx1 gene knock-out animal model based on crispr/cas9 - Google Patents

Abstract

The invention discloses a method for constructing a Glrx1 gene knock-out animal model based on CRISPR/Cas9, comprising the steps of: 1. selection and design of sgRNA targeting Glrx1 gene of mice; 2. construction of an sgRNA vector; 3. in vitro transcription of sgRNA; 4. injection of mouse fertilized eggs; 5. birth and identification of F0-generation mice; and 6. breeding of positive F0-generation mice, and birth and identification of F1-generation mice.

Description

Construction method of Glrx1 gene knockout animal model based on CRISPR/Cas9 technology Technical field

The invention belongs to the field of making a gene knockout animal model by using a genetic modification technology, and particularly relates to a method for constructing a Glrx1 gene knockout animal model based on CRISPR/Cas9 technology.

Background technique

The CRISPR/Cas (Clustered Regularly Interspaced Shot Palindromic repeats/CRISPR-associated) system is a technique for RNA-mediated Cas protein targeted modification of a gene of interest derived from bacterial acquired immunity. The Type II CRISPR/Cas9 system, which has been modified by researchers, has been successfully knocked out of mammalian cells since 2013 and has now been used for gene knockout of multiple model organisms. The CRISPR/Cas9 system vector is simple, fast, easy to operate, time-saving and labor-saving, and is suitable for almost all species. The role of CRISPR/Cas9 and TALEN (Transcription Activator-like Effector Nucleases) is to achieve double-strand breaks at specific sites on the chromosome, and then initiate autonomous damage repair, which will cause insertion or deletion, resulting in a permanent deletion of the gene sequence, ie Gene knockout. For each gene, CRISPR/Cas9 only needs to construct a single sgRNA (single guide RNA), and the efficiency is very high, the sequence selection restriction is small, only GG is needed in the genome. Compared to zinc-finger nucleases (ZFNs) and TALEN, the CRISPR/Cas9 system has the same or higher gene editing efficiency and is cheaper. Compared to TALEN, CRISPR/Cas9 caused a higher off-target effect, but the use of paired sgRNA/Cas9-D10A> truncated sgRNA or FoKI-dCas9 can greatly reduce the off-target effect. At present, CRISPR/Cas9 is mainly used for targeted site knockout of gene site-directed mutagenesis (insertion or deletion), gene-spotted knock-in, simultaneous two-point mutation, deletion of small fragments, coding genes and non-coding genes (lncRNA, microRNA). .

Glutaredoxin (Glrx) is ubiquitous in bacteria, viruses and mammals. Its expression is regulated by interferon (IFN), its molecular weight is 12kDa, it is composed of 106-107 amino acid residues, and it is thioredox. An important branch of the thioredoxin (Trx) family, as an electron donor, participates in the thiol-disulfide bond oxidoreductase family, relying on glutathione (GSH) to reduce the oxidized state protein disulfide bond to a sulfhydryl group to maintain Cell redox homeostasis plays an important role in cell signal transduction. It has been reported in the literature that in oxidative stress-induced damage, protein oxidative damage precedes nucleic acids, and proteins undergo carbonylation and glycosylation, thereby losing biological activity. Numerous studies have shown that Glrx1 is a pleiotropic cytokine with a variety of biological functions, which is closely related to the regulation of redox reaction, cell growth and inhibition of apoptosis, and certain diseases of humans, such as acquired immunodeficiency syndrome and The occurrence and development of bacterial infections are also relevant.

Glutathione is an enzyme protein that specifically and efficiently reduces glutathionylated proteins in the body. Glrx's ability to restore glutathionylated protein activity caused by oxidative stress damage may make it a hot spot. drug. Construction of the Glrx1 knockout mouse model is of great significance for the study of oxidative stress, nutritional health and so on. However, the traditional gene knockout method has a very low success rate and has not been applied. In recent years, CRISPR/Cas9 technology has been widely used, providing possibilities for the construction of Glrx1 knockout model mice and their application in nutrition and health research.

Summary of the invention

The object of the present invention is to provide a method for constructing a Glrx1 knockout animal model based on CRISPR/Cas9 technology.

The object of the present invention is achieved by the following technical solutions.

A method for constructing a Glrx1 knockout animal model based on CRISPR/Cas9 technology, comprising the following steps:

Step 1: Selection and design of gRNA targeting mouse Glrx1 gene

Designing corresponding sgRNAs at corresponding positions of the Glrx1 intron, the primer sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO.

Step 2: sgRNA vector construction

First, BsaI was digested with pUC57-sgRNA vector. After 1 h of water bath at 37 °C, 1% agarose was electrophoresed to recover the digested product; then the sgRNA primer was annealed; finally, the annealed product and the recovered digested product were ligated and transformed into E. coli. Select the monoclonal for PCR, and the PCR result is positive and sent to the sequencing to verify that the correct sgRNA vector is obtained;

Step 3: In vitro transcription of sgRNA and Cas9 mRNA using a transcription kit, and transcription of a good sgRNA for use; kit name: AM1354+AM1908, Ambion by Life Technologies;

Step 4: Microinjection of fertilized eggs of Cas9sgRNA system (Cas9 mRNA and sgRNA); Cas9 expression plasmid is cas9D10A (plasmid #42335), Addgene;

Step 5: Birth and identification of F0 generation mice;

Step 6: F0 mice were sexually matured and matured, and F1 mice were identified.

Among them, step 6 is preferred: F0 generation mice are backcrossed with C57BL/6J mice after sexual maturation, and F1 generation mice are tail-tailed at 1 week of age to obtain positive F1 heterozygotes.

It is further preferred to identify the F1 generation from mRNA levels and enzymatic sequencing.

A Glrx1 gene knockout kit based on CRISPR-Cas9 gene knockout technology, comprising:

1) an sgRNA vector comprising a pUC57-sgRNA vector as a starting vector, comprising an sgRNA targeting a Glrx1 gene; the sgRNA is annealed by an sgRNA primer represented by SEQ ID NO. 1 and SEQ ID NO. 2;

2) and supporting detection reagents for detecting the shearing effect of the Glrx1 gene and evaluating the efficiency of gene knockout.

The CRISPR-Cas9 gene knockout technology-based Glrx1 gene knockout kit of the present invention preferably further comprises Cas9 mRNA or a Cas9 expression plasmid for expressing Cas9 mRNA.

Beneficial effects:

One of the difficulties in this experiment lies in the sgRNA sequence localization. The sgRNA sequence used in this experiment is highly efficient and difficult to off target. The second is the optimization of the Cas9sgRNA system, which makes the mouse progeny more positive and the off-target rate low. The Glrx1 knockout mice produced by this technique solve the bottleneck problem of high gene off-target rate and low survival rate of animals in traditional gene knockout technology, and can be widely used in the study of dietary nutrition and health, oxidative stress and related diseases. application.

DRAWINGS

Figure 1. Glrx-1-Cas9-KO mouse strategy design

Figure 2. sgRNA vector map

Figure 3. PCR detection strategy

Figure 4, 61#, 62#, 64# electrophoresis results

Figure 5, 73 #, 74 #, 75 # electrophoresis results

Figure 6 and 7 weeks old male homozygous Glrx1 ^-/- mice photos

detailed description

Example 1

The construction method of the Glrx1 gene knockout animal model based on CRISPR/Cas9 technology is realized by the following steps:

Step 1: Selection and design of gRNA targeting mouse Glrx1 gene

The Glrx-1-Cas9-KO mouse strategy was designed as shown in Figure 1. According to the strategy, design the corresponding sgRNA sequence, according to the strategy, design the corresponding sgRNA in the corresponding position of the Glrx-1 intron, order the corresponding Oligo; sgRNA sequence is as follows:

sgRNA name	sequence	PAM
Glrx-3S1(forward)	CGGAGATGACACTTACTGATGGG (SEQ ID NO. 1)	GGG
Glrx-5S1(forward)	GCTAAGCGCCGCTGCATTACCGG (SEQ ID NO. 2)	CGG

Step 2: sgRNA vector construction

First, the pUC57-sgRNA vector was digested with BsaI, and after 1 h of water bath at 37 ° C, 1% agarose was electrophoresed, and the digested product was recovered. The ordered sgRNA primers are then annealed. Finally, the annealing product and the recovered enzyme-cut product were connected, transformed into E. coli, and the monoclonal antibody was selected for PCR. The PCR result was positive and sent to sequencing verification, and the correct sgRNA vector was obtained. The vector map is shown in FIG. 2 .

Step 3: In vitro transcription of sgRNA

The sgRNA and Cas9 mRNA were transcribed in vitro using a transcription kit, and the sgRNA and Cas9 mRNA were transcribed for later use. Kit name: AM1354+AM1908, purchased from Ambion.

Step 4: Microinjection of fertilized eggs

1. Prepare single cell fertilized eggs

Superovulation in mice: On the first day, intraperitoneal injection of horse gonadotropin gonadotropin 5IU/only, human chorionic gonadotropin injection after 46-48 hours, 2 female mice after injection of human chorionic gonadotropin Put the male mouse in the cage. On the fourth morning, the plug was checked and the score of the plug was 0.5 days.

Obtaining fertilized eggs: mice that had been sacrificed for 0.5 days after cervical vertebrae were removed, the fallopian tubes were cut out, and the mice were removed with microscopic sputum. After digestion with hyaluronidase, embryos with full morphology and uniform cytoplasm were selected and cultured in M16.

2. Microinjection of fertilized eggs

The selected fertilized eggs are transferred into the prepared M2 strips and arranged in a row (about 30-50 pieces). Place the syringe on the stage of the inverted microscope so that the strip of M2 droplets is oriented perpendicular to the operator, ie on the y-axis. The injection tube was inserted into the cytoplasm, and the Cas9sgRNA system (sgRNA and Cas9 mRNA) was injected. The Cas9 expression plasmid was cas9D10A (plasmid #42335), Addgene; and the cytoplasm was loose and the needle was quickly removed. After the end of the injection, the embryos were transferred to a Petri dish containing M16 medium and placed in a 37 ° C, 5% carbon dioxide incubator for 0.5-1.0 hours. The fertilized eggs were transplanted into the E0.5 day pseudopregnant recipient. F0 generation mice were born about 19-21 days after transplantation.

Step 5: Birth and identification of F0 generation mice

The number of birth defects was 39, and the number of surviving was 38. The F0 generation mice were identified by tail-cutting after 1 week of birth, and 7 positive F0 mice were obtained. The coat color was black, the sex was 5 females and 2 males, and Figure 6 was F1. A photo of two male homozygous Glrx1 ^-/- mice.

PCR reaction system:

Reagent	Volume (microliter)	concentration
Reaction buffer (concentrate, diluted 10 times when used)	2.5
Double distilled water	16.75
Upstream primer	1	10 micromolar
Downstream primer	1	10 micromolar
Magnesium ion (divalent)	2	25 millimoles

dNTPs	0.5	10 mmol/sample
Taq DNA polymerase	0.25	5 enzyme live units / microliter
template	1	≈100 ng / microliter

The PCR detection strategy is shown in Figure 3.

Step 6: Sexual maturity and F0 mice, F1 generation mouse identification

F0 mice were sexually matured at 8 weeks of age and C57BL/6J mice were backcrossed. The F1 mice were tail-tailed at 1 week of age, and 6 positive F1 heterozygotes were obtained. The list is as follows:

Serial number	gender	colour	genotype	male and female	Algebra
61	♂	black	-7588bp/wt, E1-E2 (the entire coding area) are deleted.	♂14	F1
62	♂	black	-7588bp/wt, E1-E2 (the entire coding area) are deleted.	♂14	F1
64	♀	black	-7588bp/wt, E1-E2 (the entire coding area) are deleted.	♂14	F1
73	♀	black	-7898bp/wt, E1-E2 (entire coding area) are all deleted	♀7	F1
74	♀	black	-7898bp/wt, E1-E2 (entire coding area) are all deleted	♀7	F1
75	♀	black	-7898bp/wt, E1-E2 (entire coding area) are all deleted	♀7	F1

The F1 generation was identified from the mRNA level and enzymatic sequencing, and the mRNA level was achieved by qPCR. The conditions were the same as above. From the sequencing results, it can be seen that the length of the 61#, 62#, 64#, 73#, 74#, 75# and wild type comparison sequences is at least -7588 bp, which means that E1-E2 is deleted.

61#,62#,64#:

73#,74#,75#:

Example 2

This example differs from Example 1 in that the single-stranded DNA template and primer sequence used in step three are 2074-Glrx-gtF1. The other steps were the same as in Example 1; the results were the same as in Example 1.

Example 3

This example differs from Example 1 in that the variety of the caged male in step 4 is preferably a C57BL/6J male. The other steps are the same as in the first embodiment.

Claims (5)

1. A method for constructing a Glrx1 knockout animal model based on CRISPR/Cas9 technology, which comprises the following steps:

   Step 1: Selection and design of sgRNA targeting mouse Glrx1 gene

   Designing corresponding sgRNAs at corresponding positions of the Glrx1 intron, the primer sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO.

   Step 2: sgRNA vector construction

   First, BsaI was digested with pUC57-sgRNA vector. After 1 h of water bath at 37 °C, 1% agarose was electrophoresed to recover the digested product; then the sgRNA primer was annealed; finally, the annealed product and the recovered digested product were ligated and transformed into E. coli. Select the monoclonal for PCR, and the PCR result is positive and sent to the sequencing to verify that the correct sgRNA vector is obtained;

   Step 3: In vitro transcription of sgRNA and Cas9 mRNA using a transcription kit, and transcription of a good sgRNA for use;

   Step 4: Microinjection of fertilized eggs of Cas9 sgRNA system consisting of Cas9 mRNA and sgRNA, wherein Cas9 expression plasmid is cas9 D10A (plasmid #42335), Addgene;

   Step 5: Birth and identification of F0 generation mice;

   Step 6: F0 mice were sexually matured and matured, and F1 mice were identified.
2. The construction method according to claim 1, characterized in that step 6: F0 generation mice are backcrossed with C57BL/6J mice after sexual maturation, and the F1 generation mice are subjected to tail-cut identification at 1 week of age. A positive F1 heterozygote was obtained.
3. The construction method according to claim 2, characterized in that the F1 generation is identified from mRNA levels and enzymatic sequencing.
4. A Glrx1 gene knockout kit based on CRISPR-Cas9 gene knockout technology, comprising:

   1) an sgRNA vector comprising a pUC57-sgRNA vector as a starting vector, comprising an sgRNA targeting a Glrx1 gene; the sgRNA is annealed by an sgRNA primer represented by SEQ ID NO. 1 and SEQ ID NO. 2;

   2) and supporting detection reagents for detecting the shearing effect of the Glrx1 gene and evaluating the efficiency of gene knockout.
5. The CRISPR-Cas9 gene knockout technology-based Glrx1 gene knockout kit according to claim 4, which further comprises Cas9 mRNA or a Cas9 expression plasmid for expressing Cas9 mRNA.

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