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Set of adenine base editors as well as related biological materials and application thereof

A technique for biomaterials, editing, applied in applications, biochemical devices and methods, plant products, etc.

Inactive Publication Date: 2020-12-29
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is how to improve the efficiency of plant adenine base editing, and how to solve and realize the base editing of the target point that the current technology cannot complete the expected adenine base editing

Method used

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  • Set of adenine base editors as well as related biological materials and application thereof
  • Set of adenine base editors as well as related biological materials and application thereof
  • Set of adenine base editors as well as related biological materials and application thereof

Examples

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Embodiment 1

[0043] Example 1. Site-directed mutation of A in the rice genome to G

[0044] 1. Construction of rice adenine base editor expression vector

[0045] This embodiment provides four kinds of rice adenine base editor expression vector pUbi-rBE of the present invention ( figure 1 ), named pUbi-rBE46b, pUbi-rBE50, pUbi-rBE54 and pUbi-rBE62, respectively. The adenine base editor expressed by pUbi-rBE46b is a fusion protein named rBE46b (also known as TadA-R-SpCas9(D10A)), composed of adenine deaminase named TadA-R and SpCas9(D10A) The Cas protein is connected with the nuclear localization signal named NLS. The amino acid sequence of rBE46b is SEQ ID No.2 in the sequence listing. In SEQ ID No.2, positions 1-167 are the amino acid sequence of TadA-R, positions 168-199 are the amino acid sequence of the connecting peptide, positions 200-1567 are the amino acid sequence of SpCas9 (D10A), and positions 1568-1576 Bit is the amino acid sequence of NLS. According to the rice codon usa...

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Abstract

The invention discloses a set of adenine base editors as well as related biological materials and application thereof. The invention provides an application of fusion protein in plant single base editing. The fusion protein has a name of TadA-R-cas and contains Cas protein and adenine deaminase, and the adenine deaminase is protein located at the 1st-167th sites and having an amino acid sequence as shown in SEQ ID No.2. The fusion protein is not only suitable for the adenine base editor containing SpCas9, but also suitable for the adenine base editor containing ScCas9, SpCas9-NG and SpRY, theapplication range of plant genome fixed-point editing is widened, and an important gene function research tool is provided for researchers in the field of plant research. The fusion protein can improve the editing efficiency and accurately mediate the mutation of a target site, and can be widely applied to rice cells.

Description

technical field [0001] The invention relates to a set of adenine base editors and related biological materials and applications in the technical field of gene editing. Background technique [0002] The CRISPR / Cas9 system is a new artificial nuclease technology, which is a complex composed of sgRNA (single guide RNA) and Cas9 protein. The genome editing technology mediated by it has become one of the most powerful tools in molecular biology , is an emerging and more precise genetic engineering technology that can modify specific target genes in the genome of organisms. Under the guidance of sgRNA, CRISPR / Cas9 causes DNA sequence-specific double-strand breaks (double-strand breaks, DSBs) at target sites in the genome through its own endonuclease activity, and then induces organisms to join ( Non-homologous end joining (NHEJ) or homologous recombination-mediated repair (homology-directed repair, HDR) in two ways. Most of the mutations induced by the NHEJ pathway are nucleotid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46C07K19/00C12N9/22C12N9/78C12N15/62
CPCC12N15/8216C12N9/22C12N9/78C12Y305/04002C07K2319/09
Inventor 周焕斌任斌严大琦柳浪严芳
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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