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Method for realizing fixed-point insertion type knockout and identification in zebra fish genome

A genomic and insertional technology, applied in the field of fixed-point insertional knockout and rapid identification, can solve the problems of random mutation, difficult positive identification, and low efficiency

Pending Publication Date: 2021-06-08
福州百维斯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method overcomes the problems of the existing random knockout methods such as low efficiency, randomness of mutations, and difficulty in positive identification.

Method used

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  • Method for realizing fixed-point insertion type knockout and identification in zebra fish genome
  • Method for realizing fixed-point insertion type knockout and identification in zebra fish genome
  • Method for realizing fixed-point insertion type knockout and identification in zebra fish genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Zebrafish Gnas gene-specific knockout

[0058] 1.1 Selection of Gnas gene sgRNA targeting sites

[0059] Exon 4 of the zebrafish Gnas gene (Gene ID: 557353) was selected for gene knockout. The sequence of exon 4 of the Gnas gene was analyzed for PAM sites on the CRISPOR website (http: / / crispor.tefor.net / crispor.py), and the PAM sites with higher scores were selected. The sequence information is as follows:

[0060] ATTGACTACATCCT (PAM) CAACTTAGCCAATCAAAAGG (sgRNA) ACTTTGAGTTC.

[0061] Gnas sgRNA sequence transcribed in vitro: GGCCUUUUGAUUGGCUAAGUUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUCACAA.

[0062] 1.2 Design of single-stranded DNA template for Gnas gene insertion knockout

[0063] According to the sgRNA target site selected in 1.1, the insertion knockout donor was designed, and the sequence information of the Gnas gene insertion knockout single-stranded DNA template was obtained as follows:

[0064] Gnas donor:...

Embodiment 2

[0091] Example 2 Zebrafish izumo1 gene-specific knockout

[0092] 1.1 Selection of izumo1 gene sgRNA targeting sites

[0093] The exon 2 of the zebrafish izumo1 gene (Gene ID: 100329365) was selected for gene knockout. The sequence of exon 2 of the izumo1 gene was analyzed for PAM sites on the CRISPOR website (http: / / crispor.tefor.net / crispor.py), and the PAM sites with higher scores were selected. The sequence information is as follows:

[0094] CAAGAACAGAATACCA (PAM)GAGTGAATTCAAGAGGCATT (sgRNA)GG.

[0095] In vitro transcribed izumo1 sgRNA sequence:

[0096] GGAAUGCCUCUUGAAUUCACUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUCACAA.

[0097] 1.2 Design of single-stranded DNA template for insertional knockout of izumo1 gene

[0098] According to the sgRNA target site selected in 1.1, the insert knockout donor was designed, and the sequence information of the izumo1 gene insert knockout single-stranded DNA template was obtained as follows:...

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Abstract

The invention provides a method for realizing fixed-point insertion type knockout and identification in a zebra fish genome. Cas9 proteins are utilized, a genome targets specific guide RNA, and a universal type translation termination sequence with short homologous arms at two ends, the middle inserted in the zebra fish genome and without similar sequences is used for assistance, so that efficient, accurate, fixed-point and directional zebrafish genome insertion type knockout is realized. Besides, a specific primer for detecting the insert fragment is provided, so that the rapid detection of gene knockout is helped to be realized. According to the method, the problems of low efficiency, mutation randomness, difficulty in positive identification and the like of an existing random knockout method are solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for realizing fixed-point insertion knockout and rapid identification in the zebrafish genome. Background technique [0002] The model organism zebrafish has high homology with human genes, and is known as the guinea pig in water. Zebrafish is an ideal model organism for developmental biology research. It has many advantages such as in vitro development, fast development, transparent embryos, and large egg production. When using zebrafish to study gene function, we need to knock out specific genes in zebrafish efficiently and quickly. In recent years, the demand for the development and application of zebrafish-based gene editing technology has become increasingly urgent. [0003] CRISPR-Cas9 is currently the most widely used system for gene knockout in zebrafish. This system has the advantages of high cutting efficiency, easy design, and low cost. However, in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N9/22C12N15/89C12Q1/686
CPCC12N15/113C12N9/22C12N15/89C12Q1/686C07K14/471C12N2310/20C12Q2565/125Y02A40/81
Inventor 郑永茂肖诗伟曾丽雯
Owner 福州百维斯生物科技有限公司
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