SgRNA, CRISPR/Cas reagent and application thereof

A technology of reagents and preparations, applied in the field of gene editing

Pending Publication Date: 2022-08-09
FUJIAN ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the use of CRISPR / RfxCas13d gene editing technology to generate a zebrafish slc25a13 gene knockdown model

Method used

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  • SgRNA, CRISPR/Cas reagent and application thereof
  • SgRNA, CRISPR/Cas reagent and application thereof
  • SgRNA, CRISPR/Cas reagent and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of CRISPR / RfxCas13d system

[0033] (1) Select the slc25a13-sgRNA targeting site on the zebrafish slc25a13 gene, and analyze the specificity of the slc25a13-sgRNA targeting site

[0034] Obtain the protein coding sequence of the zebrafish slc25a13 gene from the ZFIN database; enter the protein coding sequence of the slc25a13 gene on the CRISPOR website and design the target site, set the genome information as zebrafish, select the PAM sequence as 20bp-NGG, and select The automatically generated targeting site sequence is the sequence shown in SEQ ID NO. 12-14. Through BLAST sequence alignment, the results showed that the three slc25a13-sgRNA targeting sequences were all specific sequences of the slc25a13 gene, located in the 1st, 5th, and 14th exons of slc25a13, respectively;

[0035] (2) Preparation of slc25a13-sgRNA

[0036] 1) Design and construct the upstream primer of sgRNA: T7 promoter sequence is connected with 30nt forward repeat sequenc...

Embodiment 2

[0064] Example 2 Construction of zebrafish slc25a13 knockdown mutant model construction and verification

[0065] (1) Model construction

[0066] The RNA-Mix in Example 1 was microinjected into zygotes at the single-cell stage of zebrafish, where the final concentration of slc25a13-sgRNA was 100 ng / μL, the final concentration of mRfxCas13d was 200 ng / μL, and 10% phenol was added. Red is used to indicate whether RNA-Mix is ​​successfully introduced into zebrafish zygotes, and the liquid volume injected into each zebrafish zygote is 1nL;

[0067] (2) Identify whether the expression of the slc25a13 gene is reduced in knockdown animal models and whether the pathological phenotype of the Hyetrin deficiency syndrome appears in the model

[0068] 1) Take 10 3dpf zebrafish that have been injected successfully, add them to 1mL TRIzol reagent and transfer the mixture to a lysis matrix tube, use FastPrep-24 to homogenize twice at a rate of 5m / s, and mix the samples between the two homog...

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Abstract

The invention belongs to the technical field of gene editing, and particularly relates to an sgRNA and CRISPR / Cas reagent and application thereof, and the sgRNA and CRISPR / Cas reagent is suitable for editing zebra fish slc25a13 gene. The sgRNA comprises a sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3. The sgRNA can accurately recognize the zebra fish slc25a13 gene locus in a targeted manner, and the zebra fish slc25a13 gene can be effectively knocked out by a CRISPR / Cas technology based on the sgRNA, so that the expression of the slc25a13 gene is reduced, and the zebra fish with phenotypes related to the Citrin protein deficiency syndrome is obtained by the obtained zebra fish gene knockdown mutant under the condition of not changing genetic materials.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a sgRNA, CRISPR / Cas reagent and an application thereof. The sgRNA and CRISPR / Cas reagent are suitable for editing zebrafish slc25a13 gene. Background technique [0002] With the development of genetic engineering technology, the CRISPR / Cas family has become the most popular research tool in gene function research experiments, among which the Cas9, Cas12, and Cas13 families are the most widely used. These gene editing tools can permanently or non-permanently change genes through gene knockout or gene knockdown, link genotype to phenotype, and make forward genetics research more extensive and in-depth. RNA interference (RNAi), as a "gene knockdown" technology, is widely used in gene therapy of hereditary diseases, tumor suppression, and research on transgenic plants. , zebrafish (Danio rerio) and other teleost fish are difficult to achieve; MOs (morpholinosense ol...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N15/113C12N9/22C12N15/12C12N15/85A01K67/027
CPCC12N15/113C12N9/22C07K14/461C12N15/85A01K67/027C12N2310/20C12N2800/106A01K2227/40A01K2267/0306Y02A40/81
Inventor李丽莎林清华马丽红刘荣华卢雪花林文津
OwnerFUJIAN ACAD OF MEDICAL SCI