CRISPR/Cas9 systems and application of CRISPR/Cas9 systems in construction of alpha, beta and alpha-beta thalassemia model pig cell lines

A thalassemia and cell line technology, applied in the field of gene editing, can solve problems such as difficulty, late sexual maturity, and slow population expansion, achieve good applicability, reduce the generation of non-frameshift mutations, and improve gene editing efficiency. Effect

Active Publication Date: 2021-03-23
NANJING KGENE GENETIC ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] As far as large animals are concerned, primates are the closest relatives to humans, but they are small in size and late in sexual maturity (mating begins at 6-7 years old). And it is a single-born animal, the population multiplication speed is extremely slow, and the feeding cost is also high
In addition, primate cloning is inefficient, difficult and expensive

Method used

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  • CRISPR/Cas9 systems and application of CRISPR/Cas9 systems in construction of alpha, beta and alpha-beta thalassemia model pig cell lines
  • CRISPR/Cas9 systems and application of CRISPR/Cas9 systems in construction of alpha, beta and alpha-beta thalassemia model pig cell lines
  • CRISPR/Cas9 systems and application of CRISPR/Cas9 systems in construction of alpha, beta and alpha-beta thalassemia model pig cell lines

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Experimental program
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Effect test

Embodiment Construction

[0092] 1. Construction of Cas9 high-efficiency expression vector and detection of application effect

[0093] 1.1 Construction of Cas9 high-efficiency expression vector

[0094] The pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO vector (pKG-GE3 for short) was transformed from the addgene (Plasmid#42230, from Zhang Feng lab) pX330-U6-Chimeric_BB-CBh-hSpCas9 vector. Nuclear localization signal, adding WPRE sequence to enhance translation, adding green fluorescence and resistance screening puro gene, the map is as follows figure 1 , the base sequence is shown in SEQ ID NO:70.

[0095] The structure of the original vector pX330-U6-Chimeric_BB-CBh-hSpCas9 used is as follows figure 2 As indicated, purchased from addgene (Plasmid #42230, from Zhang Feng lab).

[0096] The build steps are as follows:

[0097] (1) Remove excess short gRNA backbone

[0098] pX330-U6-Chimeric_BB-CBh-hSpCas9( figure 2 ) was digested with BbsI and XbaI, and the vector fragment (about 8313bp) was recovered, a...

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Abstract

The invention discloses CRISPR/Cas9 systems and an application of the CRISPR/Cas9 systems in construction of alpha, beta and alpha-beta thalassemia model pig cell lines. A pair of target sequences isdesigned for each of pig HBA and HBB genes, three CRISPR/Cas9 systems are constructed by the two pairs of target sequences, each CRISPR/Cas9 system comprises a gRNA carrier and a Cas9 expression carrier, the expression carriers in the three CRISPR/Cas9 systems are respectively transferred into pig fibroblasts in proportion, and the alpha, beta and alpha-beta thalassemia model pig cell lines are obtained by screening. Single gene mutation and combined gene mutation are respectively caused to the pig HBA and HBB genes in the pig primary fibroblasts through a gene editing technology, and HBA, HBBand HBA-HBB gene mutant cells are obtained.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to the application of a gRNA target combination and a cas9 system in constructing α, β and α&β thalassemia model pig cell lines. Background technique [0002] Thalassemias (from the Greek sea, thalassa1) are a group of inherited autosomal recessive blood disorders that result in hemolytic anemia due to reduced or absent globin chain synthesis. Imbalances in the globin chains cause hemolysis, impairing erythropoiesis. About 5% of the world's population has globin variants, but only 1.7% have features of alpha or beta thalassemia. Thalassemia affects both males and females equally and occurs much less frequently in newborns, at about 4.4 per 10,000 births. Alpha thalassemia is most common in people of African and Southeast Asian descent, and beta thalassemia is most common in people of Mediterranean, African and Southeast Asian descent. Among these minorities, 5% t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N9/22C12N5/10C12N15/113
CPCC12N15/85C12N15/907C12N9/22C12N15/113C12N5/0656C12N2310/10C12N2510/00A01K2267/00C12N15/90C12N5/10
Inventor 牛冬汪滔王德华王磊程锐曾为俊马翔
Owner NANJING KGENE GENETIC ENG CO LTD
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