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Method for achieving gene knockout by utilizing base editing technique and application thereof

A gene knockout and base editing technology, applied in chemical instruments and methods, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems that the effect of primary cells and hypermethylation sites needs to be improved

Active Publication Date: 2018-11-06
李广磊
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this system can generate stop codons more efficiently, the effect on primary cells and hypermethylated sites needs to be improved

Method used

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  • Method for achieving gene knockout by utilizing base editing technique and application thereof
  • Method for achieving gene knockout by utilizing base editing technique and application thereof
  • Method for achieving gene knockout by utilizing base editing technique and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Base editing mediated by h-PHRBN-BE was performed on human cell lines, and stop codons were introduced to achieve gene knockout. Carry out gene knockout of cell lines (by electroporation or liposome transfection) according to routine operation, taking liposome transfection as an example. This embodiment selects SEQ ID NO.4-SEQ ID NO.10 as an example for illustration.

[0072] 1.1. Plasmid construction

[0073] For the selected 6 human-related gene sgRNAs, take the 20bp sequence in front of the PAM sequence and synthesize oligo, in which the upstream sequence is added with 5'-accg-3', the reverse complementary sequence is added with 5'-aaac-3', and the upstream and downstream sequences are passed Program (95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1°C / s; hold at 4°C) annealing, connected to the BsaI (NEB:R0539L) linearized pGL3-U6sgRNA (addgene: 51133) vector. The linearization system is as follows: pGL3-U6sgRNA 2μg; buffer (NEB:R0539L) 6μL; BsaI 2μL; ddH 2 O to ...

Embodiment 2

[0081] The current problem of the BE3 system is that the efficiency is low at highly methylated sites, while h-PHRBN-BE still exhibits high activity at highly methylated sites, so we chose the highly methylated FANCF site to design three sgRNAs (SEQ ID NO.11-SEQ ID NO.13), used to compare the gene knockout efficiency of BE3 and h-PHRBN-BE. Carry out gene knockout of cell lines (by electroporation or liposome transfection) according to routine operation, taking liposome transfection as an example.

[0082] 1.1. Plasmid construction

[0083]For the selected 6 human-related gene sgRNAs, take the 20bp sequence in front of the PAM sequence and synthesize oligo, in which the upstream sequence is added with 5'-accg-3', the reverse complementary sequence is added with 5'-aaac-3', and the upstream and downstream sequences are passed Program (95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1°C / s; hold at 4°C) annealing, connected to the BsaI (NEB:R0539L) linearized pGL3-U6sgRNA (addgen...

Embodiment 3

[0090] Another problem currently existing in the BE3 system is that the editing efficiency of the target site C is affected by the previous G, that is, if the G is in front of the C, the editing efficiency will be greatly reduced, but the h-PHRBN-BE system does not have such a problem, so we chose For two genes, a total of four sgRNAs (SEQ ID NO.14-SEQ ID NO.17) were designed to compare the knockout efficiency of BE3 and h-PHRBN-BE. Carry out gene knockout of cell lines (by electroporation or liposome transfection) according to routine operation, taking liposome transfection as an example.

[0091] 1.1. Plasmid construction

[0092] For the selected 6 human-related gene sgRNAs, take the 20bp sequence in front of the PAM sequence and synthesize oligo, in which the upstream sequence is added with 5'-accg-3', the reverse complementary sequence is added with 5'-aaac-3', and the upstream and downstream sequences are passed Program (95°C, 5min; 95°C-85°C at-2°C / s; 85°C-25°C at-0.1°...

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Abstract

Provided is a method for achieving gene knockout by utilizing a base editing technique and application thereof. The gene knockout method comprises the steps that a 20bp-NGG target sequence in the encoding area which genes are to be knocked out is selected and is made including a complete target codon CAA, CAG or CGA; an SgRNA sequence is utilized to localize a base editing system to the target sequence so as to change a single base C in the target codon into T, and thus a termination codon TAA, TAG or TGA is introduced to achieve gene knockout, wherein the target single base C is located at the position 3-8 of the target sequence, and the sgRNA sequence is a 20bp sequence complementary with the target sequence. The method still has higher activity in primary cells or hypermethylated regions, so the knockout method is more efficient, more accurate, less missed and wider in application range, and is a more advanced method for gene knockout.

Description

technical field [0001] The present invention relates to a gene knockout strategy, and more specifically, to a base editing-based gene knockout method and its application. Background technique [0002] With the development of high-throughput sequencing, the post-genome era has arrived. How to read the holy book of the genome is a major issue in the field of biology. The development of safer, more efficient, and more practical gene editing tools will help us understand gene function. Traditional eukaryotic targeted gene manipulation is achieved through homologous recombination of totipotent embryonic stem cells and blastocyst injection, but currently this technology is only realized in limited species such as mice. In addition, gene modification and nuclear transfer of somatic cells are also a way of gene-targeted manipulation, but there are still some defects. [0003] Programmable endonuclease technology includes zinc finger nuclease (zinc-finger nucleases, ZFNs) technolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
CPCC12N15/85C07K14/47C12N2800/80C12N2810/10
Inventor 李广磊谈方志王鑫杰
Owner 李广磊
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