Genome targeted modification method

A targeted modification and genome technology, applied in the field of genome modification, can solve the problems of inability to construct conditional knockout at one time, and inability to achieve conditional knockout at the same time.

Active Publication Date: 2019-11-22
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, gene knock-in (targeted insertion) based on NHEJ can achieve higher efficiency than HR, but this strategy cannot realize the one-time construction of conditional knock-out genes
Moreover, the existing technology cannot simultaneously achieve conditional knockout and gene / cell markers in the same embryo / individual

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199] Example 1 Creation of tbx5a conditional knockout and gene / cell marker fish lines by NHEJ-based gene knock-in

[0200] 1. Construction of tbx5a gene knock-in vector

[0201] Tbx5a is an important regulatory gene of zebrafish heart development. Based on the nucleic acid database (Nucleotide, https: / / www.ncbi.nlm.nih.gov / nucleotide / ) in the NCBI website and the zebrafish genome database in Ensembl (version Zv9, http: / / asia.ensembl.org / Danio_rerio / Info / Index) analyzed the gene structure and sequence of tbx5a. There are four transcripts of tbx5a, among which the transcripts tbx5a-001 and tbx5a-202 encode the same protein with 492aa of amino acid residues. The transcript tbx5a-201 encodes a protein of 485 aa, but this transcript was removed from the NCBI database. Transcript tbx5a-002 is a non-coding RNA. In this experiment, we chose the transcript tbx5a-202 as the main reference sequence, and its sequence is shown in SEQ ID NO:5. CRISPR / Cas target design and efficiency...

Embodiment 2

[0225] Example 2 Creation of kctd10 conditional knockout and gene / cell marker fish lines by NHEJ-based gene knock-in

[0226] A CRISPR / Cas target site was designed in the first intron of kctd10, named kctd10 I1, and its sequence is shown in SEQ ID NO:10. Efficiency was evaluated using a single restriction endonuclease Hpy188I corresponding to the target, and the results showed that the kctd10 I1 site had a high cutting activity with an efficiency close to 97%, and different forms could be detected in the target sequence by sequencing the indel. Therefore, the kctd10 I1 target was selected as the gene knock-in site for subsequent experiments ( Figure 11 ).

[0227] Based on a strategy similar to Example 1, the positive-carrier kctd10-T2A-tdGFP floxP 2PA-mutExon PoNe donor( Figure 12, hereinafter referred to as kctd10 PoNe donor), its sequence is shown in SEQ ID NO:2. Then, the kctd10 gene knock-in system was injected into single-cell stage zebrafish embryos by microinject...

Embodiment 3

[0229] Embodiment 3 prepares the genotype marker fish line of tbx5a

[0230] First, the tbx5a I2 target in Example 1 is also used as the gene knock-in site ( figure 1 ), constructed the genotype tagging vector of tbx5a tbx5a-T2A-tdT-2PA floxP tdG-2PA geno-tagging PoNe Donor( Figure 14 , hereinafter referred to as tbx5a geno-tagging donor), its sequence is shown in SEQ ID NO:3.

[0231] Inject gene knock-in system into single-cell stage zebrafish embryos by microinjection: zCas9 mRNA 300-400ng / μL, tbx5a I2 gRNA 100ng / μL, hEMX1 gRNA 100ng / μL, tbx5a geno-tagging donor(hEMX1) 15ng / μL , and obtained the F 0 Zebrafish embryos.

[0232] for the F 0 stage to verify the effectiveness of genotype markers as soon as possible. In another similar experiment, we also added Cre mRNA to the above gene knock-in system and co-injected zebrafish single-cell stage embryos. At this point, if only gene knock-in events have occurred, tdTomato in the positive element (Po-cassette) will produce ...

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Abstract

The invention relates to a genome targeted modification method. The genome targeted modification method is based on a multi-element positive and negative vector knock-in strategy and combined with a genome fixed-point modification technology to knock a positive and negative vector into atarget gene. The vector comprises a positive element and a negative element. The positive element comprises a gene coding sequence which can restore the expression the target gene, thus the function of the gene cannot be damaged, the negative element is mainly the element which destroys the function of an endogenous gene, meanwhile, an exogenous DNA sequence can be designed according to different purposes, and thus a variety of effects of fluorescent labeling and transformation, precise gene mutation, generescue and the like are achieved.

Description

[0001] This application claims the priority of the Chinese patent application with the application number 201810357493.2 and the invention title "Method for Targeted Genome Modification" submitted to the China Patent Office on April 20, 2018, the entire contents of which are incorporated herein by reference. technical field [0002] The invention relates to the field of genome modification, and more specifically relates to a method for genome targeted modification. Background technique [0003] In recent years, genome-targeted modification technologies based on artificial nucleases such as TALEN and CRISPR / Cas have been more and more widely used in basic theoretical research in life sciences, and have also shown great application potential in fields such as agriculture and medical care. At present, the application of this type of technology in the preparation of simple mutants such as indels has been relatively mature, but it is still not enough in the preparation of complex ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/65C12N15/63
CPCC12N15/65C12N15/63C12N2800/30C12N2830/36
Inventor 李文渊张博佟向军韩冰舟
Owner PEKING UNIV
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