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A method for preparing cd163 gene-edited pigs using single base editor spry-be4

A gene editing, rapobec1-xten-cas9n-ugi-nls technology, applied in the biological field, can solve the problems of PAM limitation and no research reports at the level of large animals, so as to avoid random mutation and even genome damage, improve operability and Wideness, effect of increasing freedom and broadness

Active Publication Date: 2021-03-23
北京首农未来生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology has an important defect, which is the limitation of PAM, and it is not possible to perform precise point mutations of C-T (G-A) in the target gene region at will.
[0007] In 2020, Benjamin P. Kleinstiver's research group optimized this technical defect and successfully prepared the PAM-free SpRY-BE4 technology and successfully verified it in HEK 293T cells, but there is no research report at the level of large animals

Method used

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  • A method for preparing cd163 gene-edited pigs using single base editor spry-be4
  • A method for preparing cd163 gene-edited pigs using single base editor spry-be4
  • A method for preparing cd163 gene-edited pigs using single base editor spry-be4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1. Preparation of CD163 biallelic mutant cell lines using a novel single base editor

[0060] 1. Substances for biallelic mutation of CD163

[0061] 1. Expression vector BE4-gRNA

[0062] In this study, the exon 3 region of the CD163 gene was selected as the targeting site. The sequence of the CD163 gene is shown in sequence 1, and the sequence of exon 3 is shown in sequence 2.

[0063] Three gRNAs were designed, and their recognition sequences (target sequences of sgRNAs) were: gRNA-1: TTGTCGAGGGAATGAGTCAG; gRNA-2: TCCCCATCCATCATGTTTGC; gRNA-3: GCAGTCCCAGAGAGCTGACT.

[0064] Synthesize single-stranded oligonucleotides according to the designed sequences, and the synthesis method is as follows:

[0065] gRNA-F1: CACCGTTGTCGAGGGAATGAGTCAG

[0066] gRNA-R1: AAACCTGACTCATTCCCTCGACAAC

[0067] gRNA-F2: CACCGTCCCCATCCATCATGTTTGC

[0068] gRNA-R2: AAACGCAAACATGATGGATGGGGAC

[0069] gRNA-F3: CACCGGCAGTCCCAGAGAGCTGACT

[0070] gRNA-R3: AAACAGTCAGCTCTCTGGGACTGCC ...

Embodiment 2

[0160] Embodiment 2, the preparation of CD163 biallelic knockout pig

[0161]1. Preparation of CD163 biallelic knockout pigs

[0162] 1. Take the biallelic C-T mutant fibroblasts in the logarithmic growth phase obtained in Example 1, and digest them with 0.25% trypsin for 5 minutes to obtain single cells, which will become donor cells in subsequent operations.

[0163] 2. Collect the ovaries of adult Large White pigs from the slaughterhouse, wash them three times in PBS solution at 37°C, extract follicles with a diameter of 2-8mm with a needle with a diameter of 0.7mm, and recover cumulus-oocytes with uniform shape and compact structure Cell-complexes (COCs) were washed twice with maturation solution (M199+10%FBS+0.01U / mL bFSH+0.01U / mL bLH+1µg / mL estradiol), and then the cumulus-oocyte- Put 50-60 pieces / well of the complex into a four-well plate containing maturation solution, and place it at 38.5°C, 5% CO 2 After maturing and culturing in the incubator for 18-20 hours, put ...

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Abstract

The invention discloses a method for preparing CD163 gene edited pigs by using a single base editor SpRY-BE4. The invention provides gene editing for the CD163 gene of the porcine isolated fibroblast genome, so that the 228th base of the E3 exon of the biallelic CD163 is mutated from C to T, and TGA is formed after the mutation The terminator terminates the expression of the E3 exon in advance to obtain CD163 biallelic mutant cells; the nucleotide sequence of the E3 exon is sequence 2. The CD163 double-allelic knockout pig obtained by the present invention has the ability to resist PRRSV virus, indicating that the method for preparing the CD163 double-allelic knockout pig of the present invention can obtain pigs resistant to PRRSV virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing CD163 gene-edited pigs by using a single base editor SpRY-BE4. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is a global, highly contagious and harmful porcine viral disease with different clinical symptoms in pigs of all ages, but mainly Late abortions, stillbirths and mummies in pregnant sows and respiratory disease in piglets. Its causative agent is porcine reproductive and respiratory syndrome virus (PRRSV), and this virus mainly has two types I and II. PRRSV is a single-stranded positive-sense RNA virus, a member of the order Nidovirales, the family Arteriviridae, and the genus Arterivirus. Its genome is 15kb in length and contains 11 open reading frames. The study found that the only natural host of PRRSV is pigs, which can infect differentiated blood monocytes and porcine alveolar macrophages in pigs. Calvert et al. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87C12N15/85C12N15/873C12N15/12C12N15/10C12N5/10A01K67/027
CPCA01K67/0275A01K2217/15A01K2227/108A01K2267/02C07K14/70596C12N5/0656C12N15/102C12N15/85C12N15/87C12N15/873C12N2510/00C12N2800/107
Inventor 沈秋平孙照霖李垲
Owner 北京首农未来生物科技有限公司
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