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A method for preparing precise blg gene knockout cattle using third-generation base editors

A gene knockout and editor technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of difficult point mutation technology, fragment deletion, limited HDR, etc., to avoid random Effects of mutations and even genomic damage

Active Publication Date: 2020-09-29
CHINA AGRI UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, this technology only determines the target specificity of the 20bp sequence, and there is an off-target phenomenon
Although researchers subsequently upgraded the nickase version of dCas9 and high-fidelity Cas9 on this technology, which greatly reduced the off-target effect, these technologies all use the DSB caused by the host genomic DNA, and then progressive repair through NHEJ or HDR, still exist certain defect
The efficiency of NHEJ is much higher than that of HDR. However, the repair of NHEJ is highly random, often resulting in random fragment deletion or insertion of the target gene, making it difficult to make precise point mutations. Although HDR repair has high accuracy, the efficiency of HDR repair is extremely low and Restricted to mitotic cells, limiting application of HDR repair
Therefore, accurate point mutation technology remains a big problem

Method used

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  • A method for preparing precise blg gene knockout cattle using third-generation base editors
  • A method for preparing precise blg gene knockout cattle using third-generation base editors
  • A method for preparing precise blg gene knockout cattle using third-generation base editors

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1. Preparation of BLG biallelic mutant cell lines using third-generation base editors

[0040] In this example, the third-generation base editor (BE3) was used to prepare BLG biallelic mutant cell lines. Considering the later industrialization and avoiding biological safety issues, the present invention does not use screening marker genes, but uses BE3 mRNA at the same time, so that BLG biallelic mutant cell lines without exogenous DNA integration can be prepared. For the technical route, see figure 1 . The target region sequence of BE3 single-base modification technology and the schematic diagram of BLG gene knockout achieved by single-base modification technology of BLG gene are shown in figure 2 .

[0041] According to the BLG gene sequence (SEQ ID No.1), the region where the base editing target sequence is located is located on exon 1 (SEQ ID No.2) of the BLG gene sequence, and the binding sequence of the target region is cacc c agaccatgaagggccTGG (posit...

Embodiment 2

[0088] Embodiment 2, the preparation of BLG biallelic knockout cattle

[0089] 1. Take mutant fibroblasts in the logarithmic growth phase and digest them with 0.25% trypsin for 5 minutes to obtain single cells.

[0090] 2. Collect the ovaries of adult Holstein cows 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-eggs with uniform shape and dense structure Mother cells-complexes (COCs), 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, at 38.5°C, 5% CO 2 After maturing and culturing in the incubator for 18-20 hours, put the mature oocytes into the tube of 0.1% hyaluronidase and shake for 2-3 minutes, then blow gently with a glass tube to completely separate the cumulus cells from the oo...

Embodiment 3

[0099] Example 3, Analysis of BLG protein expression in the milk of BLG biallelic knockout cows

[0100] 1. SDS-PAGE detection of BLG protein in the milk of BLG biallelic knockout cows

[0101] 1. Extract the total protein from the milk produced from the first day to the seventh day of lactation of the cow to be tested (BLG bi-allelic knockout cow or wild Holstein cow).

[0102] 2. Take the total protein of the cow to be tested and the BLG protein standard (product of Sigma) obtained in step 1, respectively, and carry out SDS-PAGE.

[0103] See the experimental results Figure 4 (M is protein marker, BLG is BLG protein standard). The results showed that the expression of BLG protein was not detected in the total protein of BLG biallelic knockout cows. It can be seen that there is no BLG protein in the milk produced by BLG biallelic knockout cows. Not only the BLG gene was knocked out at the DNA level, but also Levels are also effective in removing BLG.

[0104] 2. Western ...

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Abstract

The invention discloses a method for preparing cows with precise BLG-gene knockout by using a third-generation base editor. The method for preparing the cows with gene knockout includes the followingsteps: mutating the 277th base from the 5' end of a BLG gene in a starting cow genome into T from C, wherein the BLG gene is a DNA molecule shown in SEQ ID No. 1. A large-animal point mutation technology is established, precise single-base mutation is performed, termination codons are through exquisite design of C-T mutation so as to terminate protein translation in advance, so that the purpose ofgene knockout is achieved. Natural mutant animals are simulated, genomic double-strand break is not caused by the BE3-based point mutation technology when the method is compared with a traditional gene editing technology, and thus random mutation and even damage to the genome are avoided greatly, so that the method is safer. Important technical support is provided for researches of precise gene editing and gene knockout of cows.

Description

technical field [0001] The invention relates to a method for preparing precise BLG gene knockout cattle by using a third-generation base editor. Background technique [0002] Milk is an important nutritionally balanced healthy food consumed worldwide, including rich proteins, carbohydrates, fats and minerals. Although milk yields have been improved through years of natural breeding strategies, nutritional management and quantitative genetics, no significant changes have been achieved in milk composition. With the development of biotechnology and genetic engineering, the improvement of livestock breeds in particular provides a greater opportunity to produce "designer milk" that is especially suitable for humans. [0003] So far, various high-quality transgenic cattle have been reported, such as cattle expressing recombinant human lactoferrin, cattle with increased bovine casein content, mastitis-resistant cattle, mad cow disease-resistant cattle and their hornless cattle for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/873A01K67/027
CPCA01K67/0275A01K2217/075A01K2227/101A01K2267/02C12N15/85C12N15/873
Inventor 戴蕴平王明孙照霖丁方荣王海萍李玲
Owner CHINA AGRI UNIV
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