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Method for knocking out mouse Abhd12 gene by using CRISPR/Cas9 system and application

A systematic knockout and mouse technology, applied in the field of transgenics, to reduce the possibility of off-target and improve specificity

Pending Publication Date: 2022-07-12
CYAGEN BIOSCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no efficient method of using CRISPR / Cas9 system to knock out the mouse Abhd12 gene. It is urgent to intensify research efforts to design efficient crRNA to fill this gap

Method used

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  • Method for knocking out mouse Abhd12 gene by using CRISPR/Cas9 system and application
  • Method for knocking out mouse Abhd12 gene by using CRISPR/Cas9 system and application
  • Method for knocking out mouse Abhd12 gene by using CRISPR/Cas9 system and application

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Embodiment 1

[0035] Synthesis of targeted knockout gene sgRNA: see appendix for knockout strategy figure 1 .

[0036] The nucleotide sequence of the mouse Abhd12 gene was downloaded from the NCBI database and homologous alignment was performed. According to the PAM design principle of guideRNA and the results of homology alignment, crRNA1 and crRNA2 were synthesized, and the sequences are:

[0037] 1. Synthetic crRNA1 sequence

[0038] SEQ ID NO.1=5'-GGCACAGAGGTTCACGTTTCGUUUUAGAGCUAUGCUGUUUUG-3';

[0039] 2. Synthetic crRNA2 sequence

[0040] SEQ ID No. 2 = 5'-GTAGCACTTGTCATCTACCAGUUUUAGAGCUAUGCUGUUUUG-3'.

Embodiment 2

[0042] C57BL / 6 female mice were treated with PMSG, then injected with hCG, and mated with male mice. The fertilized eggs were collected the next day for microinjection. Item No. M0646)]: co-injected into fertilized eggs, take the fertilized eggs that survived the injection and transplant them into pseudopregnant female mice, the embryo-transferred mice will be born about 21 days after the operation, that is, the F0 generation mice. 14 days after birth, the mice were tail clipped to extract DNA and identified by PCR. The time from DNA extraction to PCR detection is 2-3 days. So this cycle takes about 40 days. The results of PCR identification of F0 mice are shown in the attachment. figure 2 .

Embodiment 3

[0044] When male Founder mice are 8 weeks old and female mice are 6 weeks old, they can be mated with wild-type heterozygous mice to obtain F1 generation heterozygous mice. The mice are identified by PCR 14 days after birth. If positive mice are born, It means that the transgene has been integrated into the germ cells, and this process takes about 120 days.

[0045] attached image 3 For the positive mouse sequencing analysis, it can be seen that 9942bp has been deleted between the genomes;

[0046] attached Figure 4 For the identification of F1 mice, mice 26, 27, 28, 35, 36 and 37 were identified as positive F1.

[0047] PCR primer F1 is designed on the upstream outside of the knockout region, R1 is designed on the downstream outside of the knockout region, and R2 is designed in the knockout region; appendix Figure 5 Relative position of PCR primers on the genome.

[0048] The primer sequences are as follows

[0049] PCR Primers1 (Annealing Temperature 60.0℃):

[0050...

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Abstract

The invention relates to the technical field of transgenosis, in particular to a method for knocking out a mouse Abhd12 gene by utilizing a CRISPR / Cas9 system and application. Comprising the following steps: (1) synthesizing crRNA aiming at an Abhd12 gene, (2) treating a C57BL / 6 female mouse by PMSG, then injecting hCG, mating with a male mouse, taking a fertilized egg in the next day, performing micro-injection, and taking out the fertilized egg; the method comprises the following steps: (1) coinjecting crRNA, tracrRNA and Cas9 protein into a fertilized egg, transplanting the fertilized egg surviving after injection into a pseudopregnant female mouse, (3) extracting DNA at the tail of an F0-generation mouse, carrying out PCR amplification, and sequencing the product, (4) respectively mating a male Founder mouse to 8 weeks old and a female mouse to 6 weeks old with a wild-type heterozygote mouse to obtain an F1-generation heterozygote mouse, and if a positive mouse is born, determining that the female mouse is born. If so, the transgene has been integrated into the germ cell. According to the invention, the target gene can be quickly and efficiently knocked out in a large fragment manner, no exogenous gene fragment is left, and the Abhd12 gene knockout mouse model can be quickly and efficiently constructed.

Description

technical field [0001] The invention relates to the field of transgenic technology, and more particularly to a method and application for knocking out the Abhd12 gene in mice by using the CRISPR / Cas9 system. Background technique [0002] The Abhd12 gene encodes an enzyme that catalyzes the hydrolysis of 2-arachidonoylglycerol (2-AG), the major endocannabinoid lipid transmitter acting on the cannabinoid receptors CB1 and CB2. The endocannabinoid system is involved in a wide range of physiological processes, including neurotransmission, mood, appetite, pain perception, addictive behaviors, and inflammation. Mutations in this gene have been linked to the neurodegenerative disease PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataracts), which are caused by inborn errors in the metabolism of endocannabinoids. Diseases associated with ABHD12 include polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract and pyramidal dystrophy. [0003...

Claims

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Application Information

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IPC IPC(8): C12N15/89C12N15/873C12N15/113A01K67/027C12N15/55A61K49/00
CPCC12N15/89C12N15/873C12N15/1137C12N9/18C12Y301/01023A01K67/0276A61K49/0008C12N2310/20A01K2227/105A01K2267/03Y02A50/30
Inventor 牟星
Owner CYAGEN BIOSCI INC
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