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Efficient clustered regularly interspaced short palindromic repeats ribonucleoprotein (CRISPR RNP) and donor DNA co-location mediated gene insertion or replacement method and application thereof

A donor and gene technology, applied in the field of genetic engineering, can solve problems affecting the accuracy and efficiency of gene insertion or replacement

Active Publication Date: 2020-08-25
AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the inconsistency of CRISPR RNP and donor DNA in time and space, that is, they do not appear at the gene editing target site at the same time, which affects the accuracy and efficiency of gene insertion or replacement.

Method used

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  • Efficient clustered regularly interspaced short palindromic repeats ribonucleoprotein (CRISPR RNP) and donor DNA co-location mediated gene insertion or replacement method and application thereof
  • Efficient clustered regularly interspaced short palindromic repeats ribonucleoprotein (CRISPR RNP) and donor DNA co-location mediated gene insertion or replacement method and application thereof
  • Efficient clustered regularly interspaced short palindromic repeats ribonucleoprotein (CRISPR RNP) and donor DNA co-location mediated gene insertion or replacement method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] (1) Preparation of Cas9-Xten-mSA protein

[0060] a. Construction of expression plasmid pET-Cas9-Xten-mSA

[0061] Synthesize the DNA (SEQ ID NO.5) sequence encoding Xten-mSA, and clone it into the pET-NLS-Cas9-NLS-6xHis vector by enzyme digestion and ligation to obtain the expression plasmid pET-Cas9-Xten-mSA; the specific steps are as follows :

[0062] The plasmid pET-NLS-Cas9-6xHis (Addgene) was purchased through Beijing Zhongyuan Company. Using this plasmid as a template, primer 5'-CGCTAGAGCTCCCGCTGCTTTTAAATATTTTG-3'(SEQ ID NO.6) and primer 5'-GCAGCTTCAACTTTTTCTTTTTGTCACTCCTAGCTGACTCAAATC-3'( SEQ ID NO.7) was amplified to obtain fragment I; using primer 5'-TGACAAAAAGAAAAAGTTGAAGCTGCATCACCACCATCACTAATG-3' (SEQ ID NO.8) and primer 5'-GCAGCCTAGGTTAATTAAGCTGCGCTAG-3' (SEQ ID NO.9) to amplify, Fragment II is obtained. Using the Overlap Extension PCR method, the mixture of fragments I and II in equimolar ratio was used as a template, and the above primers (primer SEQ ...

Embodiment 2

[0080] Example 2 Observation and Flow Cytometry Analysis of ZsGreen1 Gene Expression in Nuclear Transformed Cell Population

[0081] (1) After 24 hours of nuclear transformation in step (6) of Example 1, the expression of the ZsGreen1 gene was observed under a Zeiss Axio Observer A1 fluorescence inverted microscope, and the CRISPR RNP-donor DNA complex (sgRNA-Cas9-Xten-mSA- After ZsGreen1) nuclear transformation, about 10% of the cells showed fluorescence ( Figure 7 A); as a control, few fluorescent cells appeared after nuclear transformation of the CRISPR RNP-donor DNA hybrid (sgRNA-Cas9+ZsGreen1) ( Figure 7 B). Since the ZsGreen1 gene has no promoter when designing the donor DNA, it can only be expressed in the first intron of the Rosa26 gene through homologous recombination and depends on the promoter of the Rosa26 gene, so the fluorescent cells are correctly inserted into the ZsGreen1 gene cells at the target site. Wherein, the nuclear transformation of CRISPR RNP and...

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Abstract

The invention discloses an efficient clustered regularly interspaced short palindromic repeats ribonucleoprotein (CRISPR RNP) and donor DNA co-location mediated gene insertion or replacement method and application thereof. The method comprises the following steps: (1) biotin labeling is performed on donor DNA to obtain biotin-labeled donor DNA; (2) fusion protein of Cas9 protein and monovalent streptavidin, sgRNA and the biotin-labeled donor DNA are evenly mixed and undergo standing to obtain a CRISPR RNP-donor DNA complex; and (3) cell nucleus transformation is performed on the CRISPR RNP-donor DNA complex to realize gene insertion or replacement. According to the method, by utilizing the characteristic that the fusion protein can be combined with the biotin-labeled donor DNA, the CRISPRRNP and the donor DNA appear at a target locus together, and precise insertion of the donor DNA at the target locus or accurate replacement of a gene at the target locus is realized; and the method issuitable for the aspect of crop and livestock breeding.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an efficient CRISPR RNP and donor DNA colocalization-mediated gene insertion or replacement method and its application. Background technique [0002] In the process of CRISPR gene editing and breeding, the excellent traits of organisms are mainly achieved through three strategies: knocking out negative regulatory genes for excellent traits; single base editing and prime editing (Prime editing) to control single nucleotide changes Points and several nucleotide change sites; and homologous recombination of long regulatory sequences or coding sequences of genes with good traits in allele sites or safety sites in the genome. Gene knockout, single-base editing and guided editing, and gene insertion or replacement based on homologous recombination are the cornerstones of CRISPR gene editing technology. play a huge role. [0003] Gene knockout is to introduce t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22C07K19/00C12N15/62C12N15/70
CPCC12N15/902C12N15/907C12N15/8213C12N9/22C12N15/70C07K2319/70
Inventor 刘文华朱庆锋陈庄陈中健刘圣杰吴秀菊戴彰言
Owner AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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