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Method for increasing editing efficiency of base editing system

A base editing and editing technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of low efficiency and limitations of HDR

Inactive Publication Date: 2019-04-26
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For base-accurate substitution, the application of HDR to achieve base-accurate substitution is greatly limited due to the low efficiency of HDR and the need for DNA templates

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. High-concentration hygromycin screening improves the C→T base substitution efficiency of the target in the SpCas9n(D10A)&PmCDA1&UGI base editing system

[0043] 1. Construction of gene editing vector

[0044] The inventors of the present invention constructed the vector SpCas9n&PmCDA1&UGI.

[0045] The nucleotide sequence of the vector SpCas9n&PmCDA1&UGI (circular) is shown in sequence 1 in the sequence listing. In sequence 1, from 5' to 3', positions 131 to 467 are the nucleotide sequence of the OsU3 promoter, positions 474 to 550, positions 647 to 723 and positions 820 to 896 are all pre-tRNA Nucleotide sequence, the 551st to 570th is the nucleotide sequence of CS650, the 571st to 646th, 744th to 819th and 917th to 992th are the nucleotide sequence of the sgRNA backbone, the 724th to 743rd It is the nucleotide sequence of CS651, the 897th to 916th is the nucleotide sequence of CS652, the 993rd to 1283rd is the nucleotide sequence of the OsU3 terminator, a...

Embodiment 2

[0063] Example 2. High-concentration hygromycin screening improves the C→T base substitution efficiency of the target site by the rAPOBEC1&SpCas9n&UGI base editing system

[0064] 1. Construction of gene editing vector

[0065] The inventors of the present invention constructed the vector rAPOBEC1&SpCas9n&UGI.

[0066] The nucleotide sequence of the vector rAPOBEC1&SpCas9n&UGI (circular) is shown in sequence 2 in the sequence listing. In sequence 2, from 5' to 3', the 131st to 467th is the nucleotide sequence of the OsU3 promoter, the 474th to 550th and the 820th to 896th are all the nucleotide sequences of pre-tRNA, and the 1st Positions 551 to 570 are the nucleotide sequence of CS650, positions 571 to 646 and positions 917 to 992 are the nucleotide sequences of the sgRNA backbone, positions 897 to 916 are the nucleotide sequence of CS652, positions 993 to 1283 The position is the nucleotide sequence of the OsU3 terminator, the 1290th to 3003rd is the nucleotide sequence of...

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Abstract

The invention discloses a method for increasing editing efficiency of a base editing system. The method comprises the following steps: expressing sgRNA, anti-hygromycin protein, Cas9 protein, cytosinedeaminase and uracil DNA saccharifying enzyme inhibitor by a receptor, thereby performing base editing on a target gene in a receptor genome, wherein the receptor is a plant callus; taking hygromycinas a screening reagent for screening resistant callus during the process of introducing the plant callus, wherein the concentration of hygromycin is 35-80mg / L. An experiment proves that C arrow T base substitution efficiency of SpCas9n(D10A)&PmCDA1&UGI base editing system and rAPOBEC1&SpCas9n&UGI base editing system can be increased by increasing the concentration of screening reagent hygromycinso as to acquire an efficient mutant. The method has an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving the editing efficiency of a base editing system. 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 / Cas9protein-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 substitution is great...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/46
CPCC12N15/8213
Inventor 杨进孝张成伟徐雯袁爽李璐
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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