Fusion protein, double-deaminase-mediated base editing system containing same and application of double-deaminase-mediated base editing system

Abstract

The invention discloses a fusion protein, a double-deaminase-mediated base editing system containing the fusion protein and application of the double-deaminase-mediated base editing system. The fusion protein comprises nuclease, cytosine deaminase and adenine deaminase, and the nuclease comprises SpCas9 protein containing D10A mutation. The fusion protein and sgRNA can form a compound to form a double-deaminase-mediated base editing system, in the system, after the sgRNA and the fusion protein form the compound, the sgRNA can guide the fusion protein to recognize and cut a target sequence, and various types of base editing are generated. By using the base editing system provided by the invention, multi-type single base conversion or multi-base simultaneous conversion of C-to-G, C-to-T, C-to-A, A-to-G and the like can be efficiently realized, and random insertion or deletion of a plurality of bases can be realized. Therefore, the system can be used for constructing a gene saturation mutation library or researching a development trajectory of a specific cell group.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a fusion protein and a base editing system mediated by double deaminase containing the fusion protein and its application. Background technique [0002] Using more and more extensive high-throughput sequencing technology, people have discovered a large number of gene mutants related to human diseases or biological traits in human, animal and plant genomes, including random insertion or deletion involving multiple base mutations, Or single nucleotide variants (single nucleotide variants, SNVs). However, the relationship between these existing mutations—especially base mutations in specific gene regions—and the structure and function of genes has not yet been clearly demonstrated. To establish the connection between a specific gene and gene function, gene-specific point mutation and specific function, the most effective method is to establish a genome-wide mutation library or a saturate...

AI Technical Summary

Problems solved by technology

However, in the reported dual-base editing system, there is mutual competition between two different deaminases (cytosine deaminase and adenine deaminase) due to their different affinities for the target sequence, making The base editing efficiency of the fusion protein is lower than that of using CBE or ABE alone, and the efficiency of simultaneous target editing (C-to-T and A-to-G) of two target bases is generally below 30%.

[0005] For example, CN110835634A discloses a new type of base conversion editing system, in addition to retaining the function of a single base gene editing system—that is, realizing the conversion of a single base C-to-T at a specified site, and realizing A-to-G conversion, while It can also realize the conversion of C-to-T and A-to-G at designated sites. The new base conversion editing system includes sgRNA, nucleases capable of targeting and recognizing DNA sequences, cytosine deaminase, adenosine deaminase Ammonia and uracil glycosidase inhibitors have broken through the technical limitation of only a single type of base conversion in the prior art, and can achieve a wider range of DNA base changes, but the editing efficiency is low and the application range is small. It is difficult to meet the needs of constructing a gene saturation mutation library and building a high-throughput screening platform

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