Method for preparing cows with precise BLG-gene knockout by using third-generation base editor

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...

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

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