阅读说明：本技术 靶向小鼠Atp7b基因的gRNA及构建Wilson疾病小鼠模型的方法 (gRNA of targeted mouse Atp7b gene and method for constructing Wilson disease mouse model ) 是由 刘玉 姜伟 孙敏 于 2021-09-22 设计创作，主要内容包括：本发明属分子生物学领域,具体公开了一组靶向小鼠Atp7b基因的gRNA及构建Wilson疾病小鼠模型的方法；本发明设计了特异性靶向Atp7b基因的三条gRNA,利用cas9蛋白将Atp7b基因的Exon2-Exon20敲除,所敲区域序列为非3倍数,造成移码突变,且敲除区域包含该基因编码区的91.91%,从而达到将该基因敲除的目的；使用CRISPR/Cas9系统构建Atp7b基因敲除小鼠模型方法简单易行,周期短,拿到阳性小鼠的概率高,利用该小鼠模型可以充分研究该疾病的致病机理,并为进一步开发针对该疾病的治疗方式提供服务。(The invention belongs to the field of molecular biology, and particularly discloses a group of gRNAs targeting a mouse Atp7b gene and a method for constructing a Wilson disease mouse model; three gRNAs of specific target Atp7b gene are designed, Exon2-Exon20 of Atp7b gene is knocked out by using cas9 protein, the sequence of the knocked-out region is not 3 times, so that frame shift mutation is caused, and the knocked-out region contains 91.91% of the coding region of the gene, so that the purpose of knocking out the gene is achieved; the method for constructing the Atp7b knockout mouse model by using the CRISPR/Cas9 system is simple and easy to implement, has short period and high probability of obtaining a positive mouse, can fully research the pathogenesis of the disease by using the mouse model, and provides service for further developing a treatment mode aiming at the disease.)

1.A group of gRNAs targeting a mouse Atp7b gene is characterized by comprising three gRNAs, and the nucleic acid sequences of the three gRNAs are respectively:

gRNA1=SEQ ID NO 1=5’ -GGCACCCAGGCCAAATGTAGGGG-3’；

gRNA2=SEQ ID NO 2=5’ -GACTCTTACCACTGCTAAGTGGG-3’；

gRNA3=SEQ ID NO 3=5’ -TCTTTGCTAAGGAGTAAAACTGG-3’。

2.a gene knockout kit targeting a mouse Atp7b gene, the kit comprising a set of gRNAs targeting a mouse Atp7b gene as claimed in claim 1.

3.A method of using a set of grnas targeting a mouse BBS5 gene to construct a mouse model of Wilson's disease according to claim 1, comprising knocking out mouse Atp7b gene using gRNA1, gRNA2, gRNA 3.

4. The method of claim 3, wherein the gRNA1 is targeted to cleave the 5 'end of mouse Atp7b gene Exon2, and the gRNAs 2 and gRNA3 are targeted to cleave the 3' end of Exon 20.

5. The method according to claim 4, wherein the gRNA1, the gRNA2 and the gRNA3 excise 33183bp DNA from the 2 nd to the 20 th exons of Atp7b gene.

6. A method according to claim 3, characterized by the steps of:

1) cas9 mRNA and 3 gRNAs synthesized aiming at Atp7b genes are mixed and injected into cell nuclei of fertilized eggs with clear binuclear nuclei cultured in an M2 culture medium;

2) culturing the injected fertilized eggs in a specific culture medium, and transplanting 15-20 blastocysts into a pseudopregnant female mouse after culturing for 3.5 days to produce a mouse, namely an F0 mouse;

3) 5 days after birth of the newborn mouse, the mouse tail can be cut to extract DNA for PCR amplification identification and sequencing, and if the detection result is positive, the mouse gene knockout is successful.

7. The method of claim 6, wherein the sequencing primer in step 3 is:

PCR sequencing primer = SEQ ID NO 4= 5'-GTGGAAGAGAGGAGATGGTAAAC-3'.

8. The method of claim 6, wherein the primers used in the PCR identification in step 3 comprise:

PCR Primers1, whose sequences are:

F1=SEQ ID NO 5=5’- GTGGAAGAGAGGAGATGGTAAAC -3’；

R1= SEQ ID NO 6=5’- CATGGTTCAAGTTCACATAGTCCAG -3’。

9. the method of claim 6, wherein the primers used in the PCR identification in step 3 comprise:

PCR Primers2, whose sequences are:

F2=SEQ ID NO 7=5’- TCAGCTCAAGTGGTAAGTCCTGG -3’；

R1=SEQ ID NO 6=5’- CATGGTTCAAGTTCACATAGTCCAG -3’。

10. a Wilson disease model mouse constructed by the method of any one of claims 3 to 9.

Technical Field

The invention belongs to the field of molecular biology, and particularly discloses a group of gRNAs targeting a mouse Atp7b gene and a method for constructing a Wilson disease mouse model.

Background

Wilson's Disease (WD), also known as hepatolenticular degeneration, is a rare autosomal recessive genetic copper metabolism disorder, which is a mixed connective tissue disease among rheumatic diseases and is clinically characterized by liver diseases and neurological symptoms. The increased accumulation of copper in tissues is a major cause of liver disease, neurological disorders, the appearance of dark brown rings (kelvin-fujie) around the cornea, kidney and other organ morbidity, and tissue damage can cause bilateral softening, degeneration of the basal ganglia of the liver cirrhosis. According to the national institute of diabetes, digestive and renal diseases, about 1/10000-1/30000 people worldwide have the disease, so that the large-sample multi-center Wilson WD disease incidence investigation is not available in China at present, but the incidence of the disease in China is higher than that in the Western countries through literature analysis.

The pathogenesis of WD is the mutation of ATP7B gene, ATP7B gene is copper transport ATPase located on cell Golgi apparatus membrane, and is mainly expressed on liver cell, and about 90% of copper ions in normal human body are exported via bile mediated by ATP 7B. Normally, copper ingested by food and drink is absorbed by epithelial cells of intestinal tracts and then is transported to the hepatic sinus surface of hepatic cells through portal vein blood, and is transported to the hepatic cells through a copper transporter 1(CTR1), in the hepatic cells, copper ions are combined with a plurality of molecular chaperones and are transported to corresponding proteins to play a role, and the copper ions can be transported through antioxidant protein 1(Atox1), are transported to ATP7B molecules on Golgi apparatus, and then are combined with ceruloplasmin to play a role in secreting blood of the tract. When the ATP7B gene is mutated to cause ATP7B function to be damaged to different degrees, the copper-blue protein binding ability to transport copper ions is reduced to different degrees or completely disappears. Copper ions cannot effectively pass through bile excretion to cause accumulation in liver cells, and organelles such as mitochondria and the like are damaged in modes such as oxidative stress damage and the like, so that steatosis, necrosis and apoptosis of the liver cells are caused. Free copper ions are released into the blood after the liver cells are damaged, and other damages to the body are caused. In order to study the pathogenesis of the disease and develop an effective therapeutic method, it is essential to study the disease to construct an experimental model of the disease, and a mouse model is a disease model that is currently recognized to prepare a human disease model and to study the most effective genomic function, but a method for efficiently constructing a Wilson disease mouse model is still lacking.

Disclosure of Invention

Aiming at the defects of the prior art, the invention utilizes CRISPR/Cas9 technology to quickly knock out Atp7b gene in a mouse body and construct a WD disease model. CRISPR/Cas9 is a technology that is guided by gRNA and uses Cas9 nuclease to edit a targeted gene, and its working principle is that crRNA (CRISPR-derived RNA) is combined with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, which will guide Cas9 protein to cut double-stranded DNA at a sequence target site paired with crRNA, and through artificially designing two kinds of RNA, namely crRNA and tracrRNA, the gRNA with guiding effect is transformed, thereby guiding Cas9 protein to cut DNA at a fixed point and generate a blunt-ended double-stranded DNA gap, and further initiating a DNA damage repair mechanism, and sequences at both ends of the break are connected mainly by Non-Homologous end joining (NHEJ) or Homologous recombination (Homologous recombination, HR). If the Cas9 protein is to play a role, a 5 '-NGG-3' motif at the downstream of a target site, namely a PAM sequence, is needed, a gene-specific gRNA template sequence is positioned in front of the PAM sequence, meanwhile, more than 4T endings of the gRNA sequence are avoided, and the optimal GC content is 30-70%. In addition, the off-target effect of the gRNA is considered when the gRNA is selected, the off-target effect of the whole gene needs to consider that the number of base mismatches of an off-target site is not more than 5 as far as possible, and based on multiple considerations, three gRNAs aiming at Atp7b genes are designed in total and respectively target the 5 'end of Exon2 and the 3' end of Exon 20.

The invention comprises the following technical scheme:

a group of gRNAs targeting a mouse Atp7b gene comprises three gRNAs, and the nucleic acid sequences of the three gRNAs are respectively:

gRNA1＝SEQ ID NO 1＝5’-GGCACCCAGGCCAAATGTAGGGG-3’；

gRNA2＝SEQ ID NO 2＝5’-GACTCTTACCACTGCTAAGTGGG-3’；

gRNA3＝SEQ ID NO 3＝5’-TCTTTGCTAAGGAGTAAAACTGG-3’。

further, a gene knockout kit targeting a mouse Atp7b gene, the kit comprising the group of gRNAs targeting a mouse Atp7b gene.

Further, a method of constructing a Wilson disease mouse model using the above-described group of grnas targeting the mouse Atp7b gene, the method comprising knocking out the mouse Atp7b gene using gRNA1, gRNA2, and gRNA 3.

Further, a method of constructing a Wilson disease mouse model using the above-mentioned group of grnas targeting mouse Atp7b gene, the gRNA1 targeting cutting 5 'end of mouse Atp7b gene Exon2, and the gRNA2 and gRNA3 targeting cutting 3' end of Exon 20.

Further, a method for constructing a Wilson disease mouse model using the above-mentioned group of grnas targeting the mouse Atp7b gene, wherein the gRNA1, the gRNA2, and the gRNA3 excise a 33183bp DNA between exons 2 to 20 of the Atp7b gene.

Further, a method of constructing a Wilson disease mouse model using the above-mentioned group of grnas targeting mouse Atp7b gene, comprising the steps of:

1) cas9 mRNA and 3 gRNAs synthesized aiming at Atp7b genes are mixed and injected into cell nuclei of fertilized eggs with clear binuclear nuclei cultured in an M2 culture medium;

2) culturing the injected fertilized eggs in a specific culture medium, and transplanting 15-20 blastocysts into a pseudopregnant female mouse after culturing for 3.5 days to produce a mouse, namely an F0 mouse;

3) 5 days after birth of the newborn mouse, the mouse tail can be cut to extract DNA for PCR amplification identification and sequencing, and if the detection result is positive, the mouse gene knockout is successful.

Further, a method for constructing a Wilson disease mouse model by using the group of grnas targeting mouse Atp7b genes, wherein the sequencing primers in the step 3 are as follows:

PCR sequencing primers-SEQ ID NO 4-5'-GTGGAAGAGAGGAGATGGTAAAC-3'.

Further, a method for constructing a Wilson disease mouse model using the above-mentioned group of grnas targeting mouse Atp7b gene, wherein the primers in PCR identification in step 3 include:

PCR Primers1, whose sequences are:

F1＝SEQ ID NO 5＝5’-GTGGAAGAGAGGAGATGGTAAAC-3’；

R1＝SEQ ID NO 6＝5’-CATGGTTCAAGTTCACATAGTCCAG-3’。

further, a method for constructing a Wilson disease mouse model using the above-mentioned group of grnas targeting mouse Atp7b gene, wherein the primers in PCR identification in step 3 include:

PCR Primers2, whose sequences are:

F2＝SEQ ID NO 7＝5’-TCAGCTCAAGTGGTAAGTCCTGG-3’；

R1＝SEQ ID NO 6＝5’-CATGGTTCAAGTTCACATAGTCCAG-3’。

further, a Wilson disease model mouse obtained by a method of constructing a Wilson disease mouse model using the above-mentioned group of grnas targeting the mouse Atp7b gene.

The invention has the following beneficial effects:

the invention designs three gRNAs of specific targeting Atp7b genes, uses cas9 protein to knock out Exon2-Exon20 of Atp7b genes, the sequence of the knocked-out region is not 3 times, so that frame shift mutation is caused, and the knocked-out region comprises 91.91% of the coding region of the gene, thereby achieving the purpose of knocking out the gene. The method for constructing the Atp7b knockout mouse model by using the CRISPR/Cas9 system is simple and easy to implement, has short period and high probability of obtaining a positive mouse, can fully research the pathogenesis of the disease by using the mouse model, and provides service for further developing a treatment mode aiming at the disease.

Drawings

FIG. 1 is a schematic diagram showing a construction scheme of Atp7b knockout mouse; in the figure, a dark line segment indicates a knockout region, solid rectangle numbers represent exons of the BBS5 gene, and gRNA region represents a splicing region of gRNA;

FIG. 2.Atp7b Gene knockout mice identification strategy; the binding sites of the PCR primers used to identify the mice are shown: f1, F2, R1;

FIG. 3.Atp7 reference numeral 7b shows the results of identification of knockout mice; PCR screening is carried out by utilizing PCR Primers1, and the amplification product after gene knockout is 490 bp; 34335bp of wild type amplification product. The left figure is the DNA molecular weight marker, and the right figure is the identification product figure of PCR;

FIG. 4, Atp7, 7b genome sequencing results of knockout mice, with arrows indicating that Atp7b gene was deleted 33834bp and 11 bp.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The proper amount of the compound is determined by the ordinary technicians in the field according to the national technical specifications and the actual production conditions. The starting materials described in the present invention are all commercially available unless otherwise specified.

Examples

1. Design of knock-out protocol

According to the requirements, the detailed information of Atp7b gene is searched, the knockout region is selected to include the functional domain part of the gene as much as possible, the knockout region is selected according to the actual situation, and a Atp7b knockout region Exon2-Exon20 total 33183bp sequence which comprises 91.91% of the whole coding region of Atp7b gene is selected, as shown in the attached figure 1.

gRNA sequence design

Based on the sequence of the mouse Atp7b gene, 3 gRNA sequences for the gene were designed and synthesized, and the sequence information was as follows:

gRNA1＝SEQ ID NO 1＝5’-GGCACCCAGGCCAAATGTAGGGG-3’；

gRNA2＝SEQ ID NO 2＝5’-GACTCTTACCACTGCTAAGTGGG-3’；

gRNA3＝SEQ ID NO 3＝5’-TCTTTGCTAAGGAGTAAAACTGG-3’。

microinjection of Cas9/sgRNA

Selecting an SPF (specific pathogen free) female mouse with the age of 4-6 weeks as an ovum donor, injecting PMSG (pregnant horse serum gonadotropin) into the abdominal cavity of the mouse, injecting hCG (human chorionic gonadotropin) after 48 hours, mating the mouse with a germchit male with normal reproductive capacity, collecting fertilized ova of the mouse, digesting and washing the fertilized ova, and placing the fertilized ova in an incubator at 37 ℃ for later use. Cas9 mRNA and the artificially synthesized gRNA in experiment 2 are mixed uniformly and injected into mouse fertilized egg cell nucleus through a micro-injection method, then the mixture is transplanted into the ampulla of the oviduct of a surrogate mother mouse, the surrogate mother mouse is weighed every other week, whether the surrogate mother mouse is pregnant or not is preliminarily judged, the newborn mouse is delivered 19-21 days after the operation, and the tail number is cut and the PCR detection is carried out after the newborn mouse is delivered 5 days.

PCR identification of Atp7b knockout mice

According to Atp7b knockout region, a pair of primers F1 and R1(F1 ═ SEQ ID NO 5 ═ 5'-GTGGAAGAGAGGAGATGGTAAAC-3' R1 ═ SEQ ID NO 6 ═ 5'-CATGGTTCAAGTTCACATAGTCCAG-3') is designed at the 5 'end of Exon2 and the 3' end of Exon20 respectively, the born F0 mouse is subjected to PCR primer amplification and sequenced, the product amplified by the primers in knockout mice is 490bp, the product cannot be amplified in wild-type mice, and the identification result and the sequencing result are shown in figures 3 and 4. Mating the positive mouse and wild mouse to obtain F1 mouse, PCR amplifying to identify pure heterozygosis of mouse 14 days after birth, with the identifying primer shown in figure 2, the homozygous mouse amplification product is 490bp, the heterozygosis mouse amplification product is 490bp/502bp, and the wild mouse amplification product is 502 bp. PCR sequencing primers-SEQ ID NO 4-5'-GTGGAAGAGAGGAGATGGTAAAC-3'.

Mouse PCR identification part is rat tail, and crude cracking method is selected for rat tail identification to extract DNA.

The PCR reaction system is as follows:

components	Volume of
		ddH2O	9.0uL
F1(10um)	1.0uL
		R1(10um)	1.0uL
Premix Taq	12.5uL
		DNA	1.5uL
In all	25uL

The PCR reaction procedure was as follows:

the PCR product was subjected to agarose gel electrophoresis, and the results of the electrophoresis are shown in FIG. 3.

According to the experimental results, the following results are obtained: the invention designs three gRNAs of specific targeting Atp7b genes, uses cas9 protein to knock out Exon2-Exon20 of Atp7b genes, the sequence of the knocked-out region is not 3 times, so that frame shift mutation is caused, and the knocked-out region comprises 91.91% of the coding region of the gene, thereby achieving the purpose of knocking out the gene. The method for constructing the Atp7b knockout mouse model by using the CRISPR/Cas9 system is simple and easy to implement, has short period and high probability of obtaining a positive mouse, can fully research the pathogenesis of the disease by using the mouse model, and provides service for further developing a treatment mode aiming at the disease.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.

SEQUENCE LISTING

<110> Sai industries (Suzhou) Biotechnology Ltd

<120> gRNA targeting mouse Atp7b gene and method for constructing Wilson disease mouse model

<130> 2021

<160> 7

<170> PatentIn version 3.5

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