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A nuclear localization signal fnb and its application in improving the efficiency of base editing

A nuclear localization signal and nuclease technology, applied to nuclear localization signal FNB and its application in improving base editing efficiency, can solve the problem of low base editing efficiency, inability to be base edited, and low base editing efficiency and other problems to achieve the effect of improving the replacement efficiency

Active Publication Date: 2022-03-01
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current base editing system still has low base editing efficiency, or a target site that cannot be base edited, or a target base editing efficiency in the target site is low or cannot be edited

Method used

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  • A nuclear localization signal fnb and its application in improving the efficiency of base editing
  • A nuclear localization signal fnb and its application in improving the efficiency of base editing
  • A nuclear localization signal fnb and its application in improving the efficiency of base editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Example 1. Application of the nuclear localization signal FNB in ​​improving the efficiency of C T base substitution

[0163] 1. Design and Construction of Recombinant Expression Vectors

[0164] 1. Design of recombinant expression vector

[0165] The nuclear localization signal is added to the rAPOBEC1&Cas9n&UGI base editing system. According to the added nuclear localization signal, it is divided into four design types, and the structure diagram of the recombinant expression vector containing four different design types is as follows figure 1 shown. The design methods for the four different design types are as follows:

[0166] sCBE system: add the bpNLS nuclear localization signal before the rAPOBEC1 element in the rAPOBEC1&Cas9n&UGI base editing system, and add the bpNLS nuclear localization signal after the UGI element. The amino acid sequence of the bpNLS nuclear localization signal is as follows: KRTADGSEFEPKKKRKV (SEQ ID NO: 7). This design type is denoted a...

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Abstract

The invention discloses a nuclear localization signal FNB and its application in improving base editing efficiency. The nuclear localization signal FNB is composed of nuclear localization signal A and nuclear localization signal B, the nuclear localization signal A includes 3*Flag1 tag protein and NLS1 protein; the nuclear localization signal B includes bpNLS protein; the 3*Flag1 tag The amino acid sequence of the protein is 1-22 of sequence 8; the amino acid sequence of the NLS1 protein is 25-31 of sequence 8; the amino acid sequence of the bpNLS protein is sequence 7. It is proved by experiments that the nuclear localization signal FNB of the present invention can improve base editing efficiency, and has good application prospects in the field of biological genome editing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nuclear localization signal FNB and its application in improving base editing efficiency. Background technique [0002] CRISPR-Cas9 technology has become a powerful genome editing method and has been widely used in many tissues and cells. The CRISPR / Cas9 protein-RNA complex is positioned on the target by the guide RNA (guide RNA), cuts and generates a DNA double-strand break (dsDNA break, DSB), and then the organism will instinctively initiate a DNA repair mechanism to repair the DSB. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ), and the other is homologous recombination (homology-directed repair, HDR). Usually NHEJ accounts for the majority, so the random indels (insertions or deletions) generated by the repair are much higher than the precise repair. For precise base substitution, the application of HDR to achieve precise base ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46C12N9/22C12N9/78C07K7/08C07K19/00
CPCC12N15/8218C12N9/22C12N9/78C12Y305/04001C12Y305/04002C07K7/06C07K7/08C07K2319/43C07K2319/09
Inventor 王飞鹏杨进孝宋伟李璐袁爽
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES