GB2578026A - Method for constructing GLRX1 gene knock-out animal model based on CRISPR/CAS9 - Google Patents

Method for constructing GLRX1 gene knock-out animal model based on CRISPR/CAS9 Download PDF

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GB2578026A
GB2578026A GB1918320.1A GB201918320A GB2578026A GB 2578026 A GB2578026 A GB 2578026A GB 201918320 A GB201918320 A GB 201918320A GB 2578026 A GB2578026 A GB 2578026A
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Zhou Guanghong
Zou Xiaoyu
Shi Xuebin
Xu Xinglian
Li Chunbao
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Nanjing Agricultural University
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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

METHOD FOR CONSTRUCTING GLRX1 GENE KNOCK-OUT ANIMAL MODEL BASED ON CRISPR/CAS9
BACKGROUND
Technical Field
The present invention belongs to the field of making a gene knockout animal model by using genetic modification technology, and particularly relates to a method for constructing a Glrxl gene knock-out animal model based on CRISPR1Cas9.
Related Art A CRISPRCas (Clustered Regularly Interspaced Shot Pal indromic repeats/CR1SPR-associated) system is a technology for carrying out targeted modification on a target gene by RNA-mediated Cas protein derived from bacterial acquired immunity. Since a Type 11 CRISPR/Cas9 system modified by researchers successfully knocked out mammalian cells in 2013, it has now been used for gene knockout of multiple model organisms. The CRISPR/Cas9 system is simple and fast to construct a vector, easy to operate, time-saving, labor-saving, short in period and suitable for almost all species. The role of both CRISPR/Cas9 and TALEN (Transcription Activator-like Effector Nucleases) is to achieve double-strand breaks at specific sites on a chromosome, and then initiate autonomous damage repair, which will cause insertion or deletion, and further result in permanent deletion of gene sequences, i.e., gene knockout. For each gene, CRISPR/Cas9 only needs to construct one sgRNA (single guide RNA), the efficiency is very high, the sequence selection restriction is less, and only GO is needed in a genome. Compared to zinc-finger nucleases (ZENs) and TALEN, the CRISPR/Cas9 system has the same or higher gene editing efficiency and is cheaper. Compared to TALEN, the off-target effect caused by CRISPR/Cas9 is higher, but use of paired sgRNAICas9-D10A> truncated sgRNA or FoKI-dCas9 can greatly reduce the off-target effect. Currently, CRISPR/Cas9 is mainly used for gene-directed mutagenesis (insertion or deletion), gene-directed knock-in, simultaneous mutation of two sites, deletion of small fragments, and targeted gene knockout of coding genes and non-coding genes (lncRNA and microRNA).
Glutaredoxin (Glrx) is ubiquitous in bacteria, viruses and mammals. The expression of Glrx is regulated by interferon (IFN), and Glrx has a molecular weight of 12kDa, is composed of 106-107 amino acid residues, and is an important branch of the thioredoxin (Trx) family. As an electron donor, 6-Inc participates in composition of the sulfhydryl-disulfide bond oxidoreductase family and relies on glutathione (GSH) to reduce an oxidation-state protein disulfide bond to a sulfhydryl group, thereby maintaining cellular redox homeostasis, and playing an important role in cell signal transduction. It has been reported in 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 Glrx I is a pleiotropic cytokine with multiple biological functions, is closely related to the regulation of redox reaction, cell growth and inhibition of apoptosis, and is also associated with the occurrence and development of certain diseases of human beings, such as acquired immunodeficiency syndrome and bacterial infection.
Glrx I is a zymoprotein that can specifically and efficiently reduce glutathionylated proteins in the body. The ability of Glrx to specifically restore the activity of glutathionylated proteins generated by oxidative stress damage may make it a hot drug. Construction of a Glrxl gene knockout mouse model is of great significance for the study of oxidative stress, and nutritional health. However, a traditional gene knockout method has a very low success rate and has not been applied. In recent years, CRISPRICas9 technology has been widely used, and provides possibilities for the construction of Glrxl knockout model mice and application thereof in nutrition and health research.
SUMMARY
The present invention provides a method for constructing a Glrx I gene knockout 25 animal model based on CRISPR/Cas9 The objective of the present invention is achieved by the following technical solutions: The method for constructing the Gina gene knockout animal model based on CRISPRICas9 includes the steps of: Step 1: Selection and design of sgRNA targeting Glrxl gene of mice A corresponding sgRNA is designed in a corresponding position of a Glrx I intron, and primer sequences are shown in SEQ ID NO. I and SEQ ID NO. 2; Step 2: Construction of an sgRNA vector First, a pUC57-sgRNA vector is digested with Bsal in a water bath for 1 h at 37°C, electrophoresis is carried out on 1% agarose gel, and digested products arecollected. Then, an sgRNA primer is annealed. Finally, theannealed product and the digested product are connected.The products are transformed into Escherichia coli.Monoclones are selected for PCR, and positive PCR results are sequenced and verified to obtain the targetsgRNA vector; Step 3: In vitro transcription of sgRNA and Cas9 mRNA is carried out by using a transcription kit, and a transcribed sgRNA is for subsequent use. The kit name is AM1354+AM1908, Ambion by Life Technologies; Step 4: A Cas9 sgRNA system consisting of Cas9 mRNA and sgRNA was microinjected into fertilized eggs, wherein the Cas9 expression plasmid is cas9 D1 OA (plasm' d#42335), Addgene; Step 5: FO-generation mice are born and their genotypes areidentified; and Step 6: FO-generation mice are backcrossed, and then genotypes of F I -generation mice are identified Where, preferably in step 6, FO-generation mice are backcrossed with C57B116J mice for breeding after sexual maturation, and F! -generation mice are identified by tail-cutting at I week of age to obtain positive Fl -generation heterozygotes.
Further preferably, the Fl-generation is identified from mRNA levels and enzymatic sequencing A Glrx I gene knockout kit based on CRISPR-Cas9 gene knockout technology, including: 1) ansgRNA vector, using a pUC57-sgRNA vector as a starting vector, and comprising of sgRNA targeting Glrxl gene, wherein the sgRNA is obtained by annealingsgRNA primers as shown in SEQ ID NO. 1 and SEQ ID NO. 2; and 2) matching detection reagents, for detecting the shearing effect of the Glrx I gene and evaluating the gene knockout efficiency.
The Glrx 1 gene knockout kit based on CRISPR-Cas9 gene knockout technology provided by the present invention preferably further includes Cas9 mRNA or a Cas9 expression plasmid for expressing Cas9 mRNA.
Beneficial effects: One of the difficulties in the experiment lies in the location of sgRNA sequences. The sgRNA sequences used in the experiment are highly efficient and difficult to off target. Another difficulty lies in the optimization of a Cas9 sgRNA system, which makes the positive rate of offspring of mice higher, and the off-target rate is low. The Glrxl knockout mice produced by the technology solve the bottleneck problems of high gene off-target rate, and low survival rate of animals in traditional gene knockout technology, and can be widely used in research of dietary nutrition and health, oxidative stress and related diseases.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a design diagramfor making Gli-x-1-Cas9-K0 mouse.
Fig. 2 is ansgRNAvector.
Fig. 3 is a protocol diagram for PCR detection.
Fig. 4 is electrophoresis results of 61#, 62# and 64#.
Fig. 5 is electrophoresis results of 73#, 74# and 75#.
Fig. 6 is a photo of 7 weeks old male homozygous Glrxl mice.
DETAILED DESCRIPTION
Example 1
The method for constructing the Glncl gene knockout animal model based on CRISPR1Cas9 is realized by the following steps: Step 1: Selection and design of sgRNA targeting Glrxl gene of mice A Glrx-1-Cas9-K0 mouse policy is designed as shown in Fig. 1. A corresponding 5 sgRNA sequence is designed according to the protocol. The sgRNA in a corresponding position of a Glrx-1 intron is designed according to the protocol, and corresponding Oligo is ordered. The sgRNA sequences are as follows: sgRNA name Sequences PAM Glrx-3S1(forward) CGGAGATGACACTTACTGATGGG(SEQIDNO.1) GGG Glrx-55 I (forward) GCTA AGC GCCGCTGC A TTA CCGG(SEQIDN0.2) COG Step 2: Construction of an sgRNA vector First, a pUC57-sgRNA vector is digested with BsaI. After treatment in a water bath for 1 h at 37°C, 1% agarose electrophoresis is carried out, and a digested product is recovered.
Then, the ordered sgRNA primer is annealed. Finally, an annealed product and the recovered digested product are connected, Escherichia coli is transformed, monoclonal antibodies are selected for PCR, and positive PCR results are sequenced and verified to obtain the correct sgRNA vector. The vector map is shown in Fig. 2.
Step 3: In vitro transcription of sgRNA In vitro transcription of sgRNA and Cas9 mRNA is carried out by using a transcription kit, and transcribed sgRNA and Cas9 mRNA are for later use. The kit name is AIVI1354+AM1908, and is purchased from Ambion.
Step 4: Microinjection of fertilized eggs 1. Preparation of single cell fertilized eggs Superovulation of mice: On the first day, 5 III of horse chorionic gonadotropin is injected intraperitoneallyfor each mouse, and human chorionic gonadotropin is injected after 46-48 hours. After injection of the human chorionic gonadotropin, two female mice are caged with separated male mice. On the fourth morning, vaginal pessary is checked, and the mice with vaginal pessary are marked as 0.5 day.
Gain of the fertilized eggs: The mice with vaginal pessary for 0.5 day are sacrificed by dislocation of cervical vertebrae, fallopian tubes are cut out, and clustered eggs are taken out by microforceps. After hyaluronidase digestion, embryos with full shapes and uniform cytoplasm are selected and cultured in MI6.
2. Microinjection of fertilized eggs The selected fertilized eggs are transferred into a prepared M2 strip and arranged in a row (about 30-50 fertilized eggs). An injection dish is placed on a stage of an inverted microscope so that the M2 liquid drop strip is oriented perpendicular to an operator, i.e. on the y-axis. An injection tube is inserted into the cytoplasm to inject the Cas9 sgRNA system (sgRNA and Cas9 mRNA), and the Cas9 expression plasmid is cas9D 1 OA (plasmid 1412335), Addgene. The needle is withdrawn quickly when the cytoplasm is loose. After the injection, the embryos are transferred to a petri dish containing MI 6 culture medium and placed in a 37°C incubator containing 5% carbon dioxide for restoring for 0.5-1.0 hour. The fertilized eggs are transplanted into Ea 5-day pseudopregnant receptors. FO-generation mice are born about 19-21 days after transplantation.
Step 5: Birth and identification of FO-generation mice The number of births is 39, and the number of live births is 38. The FO-generation mice are identified by tail-cutting after I week of birth, and 7 positive FO-generation mice are obtained, including 5 females and 2 males. The coat color is black. Fig. 6 is a photo of two Fl-generation male homozygous Glrx1-1-mice.
PCR reaction system: Reagents Volume (microliter) Concentration Reaction buffer (stock, diluted 10 times when 2.5 used) Double distilled water 16.75 Upstream primer 1 10 micromoles Downstream primer 1 10 micromoles Magnesium ion (divalent) 2 25 millimoles dNTPs 0.5 10 millimoles/specimen TaqDNApolymerase 0.25 5 U/I1L Template 1 about 10Ong/tIL The PCR detection protocol is shown in Fig. 3.
Serial number Primer name Primer sequence Amplified fragment length 1 2074-61rx-01 GTGGCAAAGTTCAGTCACAA Wild type-8603bp Gene knock-out: about lkb 2074-G1rx-gtR I TCCTCTTCTGGGCAACTGTC Conventional PC R procedure Touchdown PCR procedure Primer used: Primer used: I Step Temperature Time Circulation Step Temperature Time Circulation +Temperature.
/Circulation I 95°C 5 min 1 95°C 5 liii 11 2 95°C 30s 2 98°C 30s 3 58°C 30 s 3 65°C 30s -0.5 4 72°C 45s 2-4,35x 4 72°C 45s 2-4,20x 72°C 5 min 5 98°C 30 s 6 10°C Holding 6 55°C 30 s 72°C 45 s 5 20x 8 72°C 5 min 9 10°C Holding Step 6: Sex-maturity breeding of FO-generation mice and identification of F I -generation mice FO-generation mice are backcrossed with C57B1_16J mice for breeding after sexual 5 maturation at about 8 weeks of age. Fl-generation mice are identified by tail-cutting at I week of age to obtain 6 positive Fl-generation heterozygotes listed as follows: Serial Gender Color Genotype Female/male Generation number 61 Black -7588bp/wt, EI-E2(cntirc coding area) all deleted 14 Fl 62 H Black -7588bp/wt, El-E2 (entire coding area) all deleted a'14 Fl 64 Black -7588bp/wt, El-E2 (entire coding area) all deleted (:.,14 Fl 73 Black -7898bp/wk E1-E2 (entire coding area) all deleted 27 Fl 74 4: Black -7898bp/wk El-E2 (entire coding area) all deleted 27 Fl 4: Black -7898bp/wk E1-E2 (entire coding area) all deleted 27 Fl The Fl-generation is identified from the mRNA level and enzymatic sequencing. The mRNA level is quantifiedby qPCR under the same conditions as above. From the sequencing results, the length of sequences of the 614, 624, 644, 734, 74 and 75# is at least -7588 bp less than that of a wild type, which means that E1-E2 is deleted.
61#, 62#, 64#:
GCCCTTTAAAACTGAAGCATCCTACTTGGTAACTCCTCCTCCAAGGAGGTTCCTTATTAAA
TGACiAGCTCiCTGGCIAAGCOCC 7588bp ACACATACiTTC FACTACAIAA ATACACA/AAAAGATAACGT 73#, 74#, 75#: CCAGTGTGCA ATGGTAGGCCTAGGAAGTA CTGACTC ATACC AA 78981w
AGCTAAGGATGGAAATTTGGGAAGTAT
Sample 2 The present sample differs from Sample 1 in that the single-stranded DNA template and primer sequence used in step 3 are 2074-Glrx-gtF I The other steps are the same as those in Samplel, and the results are the same as those in Sample 1.
Sample3 The present samplediffers from Sample 1 in that the variety of the caged male mice in step 4 is preferably C57BLI6J male mice. The other steps are the same as those in Sample].

Claims (5)

  1. CLAIMSWhat is claimed is: 1. A method for constructing a Glrxl gene knock-out animal model based on CR1SPRitas9, comprising of the following steps: Stcp 1: Selection and design of sgRNA targeting Gina gene of mice AcorrespondingsgRNA is designed in acorresponding position of a Glrx I intron, and primer sequences thereof are shown in SEQ ID NO. 1 and SEQ ID NO. 2; Step 2: Construction of an sgRNA vector First, a pUC57-sgRNA vector is digested with Bsal in a water bathfor 1 h at 37°C,thenelectrophoresis is carried out on 1% agarose,thedigested product is collected, then, ansgRNA primer is annealed,theannealed products and the digested products are connected,the PCR product istransformed intoEscherichia coli, monoclones are selected for PCR, and positive PCR results are sequenced and verified to obtain the targetsgRNA vector; Step 3: In vitro transcription of sgRNA and Cas9 mRNA is carried out by using a transcription kit, and atranscribed sgRNA is for subsequent use; Step 4:A Cas9 sgRNA system consisting of Cas9 mRNA and sgRNAwas microinjectedinto fertilized eggs, whereintheCas9 expression plasmid is cas9 D1 OA (plasmid#42335), Addgene; Step 5:F0-generation mice are born and their genotypes are identified: and Step 6:F0-generation mice are backcrossed, and then genotypes of F I -generation mice are identified.
  2. 2. The construction method according to claim 1, wherein in step 6, FO-generation mice are backcrossed with C57B1J6J mice for breeding after sexual maturation, and Fl -generation mice are identified by tail-cutting at I week of age to obtain positive F I -generation heterozygotes.
  3. 3. The construction method according to claim 2, wherein Fl -generation is identified from the mRNA level and enzymatic sequencing
  4. 4. A Glrxl gene knockout kit based on CRISPR-Cas9 gene knockout technology, comprising of: 1) ansgRNA vector, using a pUC57-sgRNA vector as a starting vector, and comprising of sgRNA targeting Glrx I gene, wherein the sgRNA is obtained by annealingsgRNA primers as shown in SEQ ID NO I and SEQ ID NO. 2; and 2) matching detection reagents, for detecting the shearing effect of the Glrxl gene and evaluating the gene knockout efficiency.
  5. 5. The Glrxl gene knockout kit based on CRISPR-Cas9 gene knockout technology according to claim 4, wherein the kit further comprises of Cas9 mRNA or a Cas9 expression plasmid for expressing Cas9 mRNA.
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US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9322037B2 (en) 2013-09-06 2016-04-26 President And Fellows Of Harvard College Cas9-FokI fusion proteins and uses thereof
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US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
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