Nuclear localization signal F4NLS and application thereof in improvement of basic group editing efficiency and expansion of editable basic group range

A nuclear localization signal and encoding technology, applied in the biological field, can solve problems such as inability to be edited by bases, low base editing efficiency, and inability to be edited

Active Publication Date: 2020-04-03
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether it is a CBE system or an ABE system, there are still situations where base editing efficiency is not high, or a target that cannot be base-edited, or a target C or A within a target has low editing efficiency or cannot be edited

Method used

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  • Nuclear localization signal F4NLS and application thereof in improvement of basic group editing efficiency and expansion of editable basic group range
  • Nuclear localization signal F4NLS and application thereof in improvement of basic group editing efficiency and expansion of editable basic group range
  • Nuclear localization signal F4NLS and application thereof in improvement of basic group editing efficiency and expansion of editable basic group range

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0213] Example 1. Application of the nuclear localization signal F4NLS in improving the efficiency of A·G base substitution or expanding the range of editable A

[0214] 1. Design and Construction of Recombinant Expression Vectors

[0215] 1. Design of recombinant expression vector

[0216] The nuclear localization signal is added to the ecTadA&ecTadA*&Cas9n base editing system. According to the added nuclear localization signal, it is divided into three design types, and the structure diagram of the recombinant expression vector containing three different design types is as follows: figure 1 shown. The design methods for the three different design types are as follows:

[0217] sABE system: add the bpNLS nuclear localization signal before the ecTadA element in the ecTadA&ecTadA*&Cas9n base editing system, and add the bpNLS nuclear localization signal after the Cas9n element. This design type is denoted as bpNLS-bpNLS. The amino acid sequence of the bpNLS nuclear localizat...

Embodiment 2

[0253] Example 2, the application of the nuclear localization signal F4NLS in improving the efficiency of C T base substitution

[0254] 1. Design and Construction of Recombinant Expression Vectors

[0255] 1. Design of recombinant expression vector

[0256] 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 two design types. The structure diagram of the recombinant expression vector containing two different design types is as follows: figure 2 shown. The design methods for the two different design types are as follows:

[0257] 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.

[0258] F4NLS-sCBE system: Add 3*Flag&4*NLS nuclear localization signal before the rAPOBEC1 element in the rAPOBEC1&Cas9n&UGI base editing syste...

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Abstract

The invention discloses a nuclear localization signal F4NLS and application thereof in improvement of basic group editing efficiency and expansion of an editable basic group range. The nuclear localization signal F4NLS is composed of a nuclear localization signal A and a nuclear localization signal B: the nuclear localization signal A comprises 3*Flag tag protein and NLS protein; and the nuclear localization signal B comprises NLS protein. The amino acid sequence of the 3*Flag tag protein is the 1st to 22nd sites of a sequence 10; the amino acid sequence of the NLS protein is the 1st to 7th sites of a sequence 11. Experiments prove that the nuclear localization signal F4NLS can improve the basic group editing efficiency and expand the editable basic group range, and has a good applicationprospect 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 F4NLS and its application in improving base editing efficiency and expanding the range of editable bases. 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...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/29C12N9/78C12N15/55C12N15/82C12N5/10C07K14/415A01H5/00A01H6/46
CPCC12N15/113C12N9/22C12N9/78C12N15/8218C07K14/415C12Y305/04001C12Y305/04002
Inventor 杨进孝王飞鹏赵久然张成伟徐雯
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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