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CRISPR/Cas9 system mediated method for goat KRTAP13-1 gene knockout

A system-guided, goat technology, applied in the fields of molecular biology and animal genetics and breeding, can solve the problems of no research revealing the KRTAP13-1 gene and no reports, and achieve the effect of improving the efficiency of gene editing and improving the safety.

Pending Publication Date: 2019-08-27
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no research has revealed the specific mechanism of the KRTAP13-1 gene on the structure and development of hair follicles, so it is of great significance to explore the KRTAP13-1 gene on hair follicle research.
[0005] Knockout of goat KRTAP13-1 gene using CRISPER-Cas9 system has not been reported

Method used

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  • CRISPR/Cas9 system mediated method for goat KRTAP13-1 gene knockout
  • CRISPR/Cas9 system mediated method for goat KRTAP13-1 gene knockout
  • CRISPR/Cas9 system mediated method for goat KRTAP13-1 gene knockout

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Optimization of Cas9-gRNA expression vector

[0033] The Cas9 / gRNA co-expression vector purchased from Beijing Weishang Lide Biotechnology Co., Ltd. was optimized. The nucleotide sequence of the optimized Cas9 / gRNA co-expression vector is shown in SEQ ID NO: 1, and the plasmid map is shown in figure 1 .

Embodiment 2

[0034] Example 2 Construction of Cas9 / gRNA co-expression vector

[0035] According to the goat KRTAP13-1 gene sequence (Gene ID: 102181431), the gene sequence is shown in SEQ ID NO:2. The gRNA sequence was designed on the exon sequence of the KRTAP13-1 gene, and a Cas9 / gRNA co-expression vector based on the CRISPER / Cas9 system was constructed. Utilize biological software to design the sgRNA sequence according to the sgRNA action site, and the sequence is shown in SEQ ID NO:3. Synthesize Target-Sense and Target-Anti, whose sequences are shown in SEQ ID NO: 4, 5, anneal to form a partially complementary oligo dimer, and then insert the oligo dimer into the Cas9 / gRNA expression vector to obtain a complete The Cas9 / gRNA co-expression vector was transformed into Escherichia coli Trans1-T1, plated, and after 9 hours, a single colony was picked, shaken, and the bacterial liquid was identified by PCR. A single colony with correct sequencing was inoculated in LB medium containing Amp...

Embodiment 3

[0036] Example 3 Electroporation transfection of goat fetal fibroblasts

[0037] When the confluence of the cells reaches 80% of the bottom of the 100mm flat dish, the number of cells and the activity state are ideal, and the cell electrotransfection is performed. First slowly remove the cell culture waste liquid along the wall of the plate, slowly add PBS along the wall of the plate and discard it, use 2mL trypsin to digest the cells until they are round in shape but have not yet floated from the bottom of the plate, use 5mL containing 10 %FBS in culture medium to stop the digestion process. After washing the bottom of the dish repeatedly with a pipette, the cell suspension was collected and centrifuged at 1500rpm for 5min, then the supernatant was removed. After washing the cell pellet three times with opti-MEM electroporation buffer, collect the cell pellet and resuspend in 80 μL opti-MEM, add 2 μg of Cas9 / gRNA co-expression vector plasmid, make up the volume to 100 μL wit...

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Abstract

The invention uses a CRISPR / Cas9 system to mediate completion of fixed-point knockout of a goat KRTAP13-1 gene. According to a KRTAP13-1 gene sequence of a goat, a Cas9 / gRNA targeting vector based onthe CRISPR / Cas9 system is constructed by the research; an optimized Cas9 / gRNA coexpression knockout vector is transfected into a goat fetus fibroblast to obtain a KRTAP13-1 gene fixed-point knockout cell strain. The knockout vector constructed by the invention based on the CRISPR / Cas9 system provides a simple, fast and safe approach for the fixed-point knockout of the goat KRTAP13-1 gene. The method does not involve any selective marker gene in a cell strain screening process, thereby greatly improving safety of transgenic animals, and has important value to the study of the genetic function and genetic breeding of the goat.

Description

technical field [0001] The invention relates to the fields of molecular biology and animal genetic breeding, in particular to a method for targeted knockout of goat KRTAP13-1 gene mediated by CRISPR / Cas9 technology. Background technique [0002] In the current field of life sciences, the CRISPR / Cas system has shown great potential and unprecedented influence as a third-generation gene editing tool. It has caused great changes in the fields of zoology, botany and medicine, and has also been fully affirmed. Before the emergence of the CRISPR / Cas system, the site-directed modification of genes was mainly achieved by zinc finger nucleases (ZFNs) and transcriptional activator effectors (TALEs). However, these two techniques are limited to certain model organisms and have low efficiency. They also require complex protein engineering for each target site, which greatly increases the difficulty and cost of experiments. The CRISPR / Cas9 system is different from the first-generation ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N5/10C12N15/877A01K67/027
CPCC12N15/85C12N15/907C12N5/0603C12N5/0656C12N15/8772C07K14/4741A01K67/0276C12N2800/107C12N2810/10C12N2510/00A01K2217/075A01K2227/102
Inventor 刘东军高源郝斐呼啸多磊
Owner INNER MONGOLIA UNIVERSITY
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