CRISPR/Cas9-gRNA targeting sequence pair, plasmid and HD cell model of HTT

A cas9-gRNA, cell model technology, applied in the field of plasmids and HD cell models, can solve problems such as practical application limitations, and achieve the effect of good gene editing activity

Active Publication Date: 2018-10-23
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And because HD is a serious neurological disease symptom that occurs after a large amount of chronic mutated protein accumulates, it takes a long time for the onset of mutated protein to accumulate and patients generally have a survival period of 15-20 years after the onset, so the HD of iPSCs and ESCs Practical applications of cell models are limited

Method used

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  • CRISPR/Cas9-gRNA targeting sequence pair, plasmid and HD cell model of HTT
  • CRISPR/Cas9-gRNA targeting sequence pair, plasmid and HD cell model of HTT
  • CRISPR/Cas9-gRNA targeting sequence pair, plasmid and HD cell model of HTT

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] According to GenBank collection of mouse (C57BL / 6J) HTT gene information:

[0038] Description: huntingtin[Source:MGI Symbol; Acc:MGI:96067]

[0039] Synonyms: IT15,C430023I11Rik,Hdh,HD,htt,huntingtin

[0040] Location: Chromosome 5:34,761,740-34,912,534forward strand.

[0041] Gene ID: 15194

[0042] And comprehensively analyze the HTT genome structure and protein function conserved region, design CRISPR target (gRNA), the target sequence is as follows:

[0043] HTT-L7: tccgccggcatgacgtcacggg;

[0044] HTT-L8: ccgcgagggttgccgggacgg;

[0045] HTT-L9: caagatggctgagcgccttgg;

[0046] HTT-L10:cattgccttgctgctaagtgg;

[0047] HTT-R7: ttggccatgcccagcacgcagg;

[0048] reverse complement: cctgcgtgctgggcatggccaa;

[0049] HTT-R8: taccgcgaccctctggacaggg;

[0050] reverse complement: ccctgtccagagggtcgcggta;

[0051] HTT-R9: cgggaaagcctggcctcaggg;

[0052] reverse complement: ccctgaggccaggctttcccg;

[0053] HTT-R10: cggctctgtctcctctgaggg;

[0054] Reverse complement: c...

Embodiment 2

[0090] This example provides the CRISPR / Cas9-gRNA targeting plasmid of HTT and its construction method. The targeting plasmid is obtained by constructing a pair of targeting sequences into a carrier plasmid. The targeting sequence pair is composed of L sequence and R sequence, so that the sense strand and antisense strand of the target gene (dsDNA) can be cut at the same time, so that the target gene can be knocked into the target site by HR. According to the in vitro activity detection results in Example 1, the activities of the Htt-L9 and Htt-R9 targets are both about 95%, indicating that the targeting plasmid composed of the Htt-L9 and Htt-R9 target pairing has a relatively high in vivo gene editing activity , so in this example, Htt-L9 and Htt-R9 were selected to form a targeting sequence pair, and the targeting sequence pair was constructed into a vector plasmid. The carrier plasmid is a carrier plasmid suitable for mammals and / or mammalian cells. In order to facilitate t...

Embodiment 3

[0094] In this example, the NQ (150Q, 90Q, 50Q, 20Q) Donor plasmid was designed and constructed by Gibson cloning based on the nucleotide sequences and relative positions of the L and R targets of the Htt9 plasmid. Schematic diagram of Donor plasmid design and construction image 3 shown. image 3 In the figure, the restriction sites between PCR fragment 4 and PCR fragments 1 and 3 are Not1 restriction sites (represented in yellow in the original figure), and the restriction sites between PCR fragment 1 and PCR fragment 2 are EcoR1 and Xma1 restriction sites point (represented in red in the original figure), between PCR fragment 2 and PCR fragment 3 is the FRT (directed recombinase recognition site) site (represented in blue in the original figure).

[0095] 1. Extract CATH-a cell genomic DNA

[0096] CATH-a (ATCC CRL-11179) was purchased from Beijing Bena Chuanglian Biotechnology Research Institute and was frozen for this experiment. The cryopreserved cells were resuscitat...

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Abstract

The invention discloses a CRISPR / Cas9-gRNA targeting sequence pair, a plasmid and an HD model of HTT, and relates to the field of biotechnology. The targeting sequence pair of the invention comprisesan L sequence and an R sequence. The base sequence of the L sequence is shown as SEQ ID NO.1. The base sequence of the R sequence is shown as SEQ ID NO.2. The targeting plasmid of the invention comprises a first vector plasmid and the above-mentioned targeting sequence pair. The targeting sequence pair is constructed into the first vector plasmid. The HD cell model of the invention is obtained byco-transfecting cells with PolyQ Donor plasmids and the above-mentioned targeting plasmids. By using differentiated neuron cells as a carrier to construct the HD cell model, the invention provides a research platform for exploring the influences or changes of mHtt protein on the internal environment of differentiated nerve cells, and studying the changes of various signal pathways, metabolic pathways and intracellular homeostasis caused by mHtt protein in differentiated neuronal cells.

Description

technical field [0001] The invention relates to the field of biotechnology, especially the CRISPR / Cas9-gRNA targeting sequence pair, plasmid and HD cell model of HTT. Background technique [0002] Huntington's disease (HD) is an autosomal dominant genetic disease, the main focus of which is in the brain, and its exact pathogenesis is unclear. Clinically, HD is mainly manifested as progressive motor, cognitive and mental disorders, but most of the final causes of death of patients are caused by complications. At present, there are no effective drugs and methods for treating HD, and the treatment for HD mainly focuses on relieving symptoms. In the early stage of the disease, when the symptoms are not enough to affect the patient's daily life, drug intervention is usually not needed. When a patient falls, drug intervention should be considered. The first choice is a dopamine-depleting drug: tetrabenazine, although it may completely inhibit excessive movement and can effective...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N5/10C12N15/85
CPCC12N5/0619C12N15/85C12N2510/00C12N2800/80C12N2810/10
Inventor 付彬徐兴然彭怡王柏彬杨春丽吴惠杨丹
Owner SOUTHWEST UNIV
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