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XCas9n-epBE base editing system and application thereof in genome base substitution

A genome and editing technology, applied in the direction of application, genetic engineering, other methods of inserting foreign genetic material, etc.

Active Publication Date: 2019-12-10
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports of C-to-T base editing of GAC and GAG PAM targets in either animal or plant cells

Method used

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  • XCas9n-epBE base editing system and application thereof in genome base substitution
  • XCas9n-epBE base editing system and application thereof in genome base substitution

Examples

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Comparison scheme
Effect test

Embodiment 1

[0133] Example 1. Construction of the xCas9n-epBE base editing system vector and its application in the base substitution of the PAM sequence as the target of GAC and GAG in the rice genome

[0134] 1. Construction of xCas9n-epBE base editing system vector

[0135] Artificially synthesize the following recombinant expression vectors, each of which is a circular plasmid: xCas9n-epBE-1 recombinant expression vector and xCas9n-epBE-2 recombinant expression vector. Schematic diagram of each element structure of xCas9n-epBE-1 recombinant expression vector and xCas9n-epBE-2 recombinant expression vector figure 1 shown. The cytidine deaminase is PmCDA1, the sgRNA backbone is esgRNA, and the multi-target system uses the tRNA system. The specific structure description is as follows:

[0136]The sequence of the xCas9n-epBE-1 recombinant expression vector is sequence 1 in the sequence list. The 131-467th position of sequence 1 is the nucleotide sequence of the OsU3 promoter, the 474...

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Abstract

The invention discloses an xCas9n-epBE base editing system and an application thereof in genome base substitution. The xCas9n-epBE base editing system comprises xCas9n, PmCDA1, UGI and tRNA-esgRNA; tRNA-esgRNA targets a target sequence; and tRNA-esgRNA is represented as formula I shown as a tRNA-target sequence transcripted RNA-esgRNA skeleton. Experiments prove that the xCas9n-epBE base editing system can realize target sequence editing in plant genomes, and particularly realize the base substitution from base C to base T in the target sequence when the PAM sequence is GAC or GAG. The xCas9n-epBE base editing system has broad application prospects in the base substitution in plant or animal genomes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an xCas9n-epBE base editing system and its application in genome base replacement. 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 substitution is great...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/90C12N9/22C07K19/00A01H5/00A01H6/46
CPCC12N15/8213C12N15/902C12N9/22C12N15/8205C07K2319/00
Inventor 杨进孝张成伟徐雯吕欣欣李璐
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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